Abstract: Nile tilapia (Oreochromis niloticus) is one of the commercially important fish species in the southern China, but the lack of cold-resistant strains in tilapia results in a large number of deaths in winter and early spring. Some heat-shock proteins (Hsps) have been revealed to facilitate correct folding of other proteins under diverse forms of physiological stresses, such as extreme temperatures, chemical toxicity and oxidative stress. To understand the molecular mechanism of cold tolerance in fish, we cloned the full-length cDNAs of TCP-1-beta and TCP-1-eta of the Hsp60 family in nile tilapia (Oreochromis niloticus), and studied their expression patterns under normal and low temperature treatments. The TCP-1-beta cDNA consisted of 1755 bp and encoded a peptide of 534 amino acids. The TCP-1-eta cDNA consisted of 1651 bp and encoded a peptide of 545 amino acids. Multiple alignment and homological analysis revealed their evolutionary conservation and functional significance of TCP-1-beta and TCP-1-eta among eukaryotes. Significantly, the deduced amino acid sequences of TCP-1-beta and TCP-1-eta contained highly conserved amino-acid motifs and three ATP-binding sites. The tilapia TCP-1-beta was highly homologous with TCP-1-beta in other species, sharing 92% identities with Salmo salar and Danio rerio, 89% with Xenopus laevis, 88% with Mus musculus, 87% with Homo sapiens, and 73% with Drosophila melanogaster. Tilapia TCP-1-eta shared 92% identities with that of Danio rerio, 89% with Xenopus laevis, 88% with Mus musculus, 89% with Homo sapiens, and 76% with Drosophila melanogaster. Phylogenetic trees were also constructed among amino acid sequences of TCP-1-beta and TCP-1-eta in eukaryotes. The tilapia TCP-1-beta was clustered with Notothenia coriiceps TCP-1-beta, and tilapia TCP-1-eta was clustered with Danio rerio TCP-1-eta. Total RNAs were extracted from seven tissues including spleen, gut, kidney, heart, brain, muscle and hepatopancreas. Using beta-actin as internal control, their expression patterns were analyzed by real-time PCR, in which TCP-1-beta and TCP-1-eta mRNA existed in all seven tissues, and expressed mainly in muscle. To study the response of TCP-1 to low temperatures, the mRNA levels of TCP-1-beta and TCP-1-eta were analyzed in response to cold induction treatments. The experimental fishes were divided into five groups and kept at different temperatures (22℃, 18℃, 16℃, 14℃ and 12℃) for 48 hours. Total RNAs were extracted from hepatopancreas tissue at different temperature treatments (22℃, 18℃, 16℃, 14℃ and 12℃). Real-time PCR analysis revealed that TCP-1-beta and TCP-1-eta mRNA increased at 18℃ and peaked at 12℃. TCP-1-beta mRNA was 2.8 fold up-regulated at 18℃ compared to normal temperature at 22℃, and then held stable until the induction temperature changed to 12℃ which induced 12.2 fold increase in mRNA levels of TCP-1-beta. The expression level of TCP-1-eta was 2 fold upregulated at 18℃ compared to normal temperature at 22℃, and then a gradual increase in the level of TCP-1-eta was observed, reaching a peak of 10.7 fold increase at 12℃. The cold-induced expression of TCP-1-beta and TCP-1-eta suggested that they might be cold tolerance-related genes in Nile tilapia. Therefore, this study established a basis for studying the molecular mechanism of cold tolerance in Nile tilapia.