Here, we report the identification and characterization of a new hsp40 family member, heat shock cognate 40 (hsc40), which may be a specific functional partner for hsc70. The hsc40 gene consists of five exons and four introns. The previously characterized hsp40 gene contains only three exons and two introns. Despite this difference in the numbers of exons and introns, the structures of these two genes are actually quite similar. If the first two exons and introns of the hsc40 gene are excluded, the rest is essentially the same as the whole hsp40 gene. The intron/exon boundaries of the hsc40 and hsp40 genes are conserved with respect to amino acid coding, indicating that the hsp40 gene might have arisen from a duplicated hsc40 gene, which developed a new transcription-regulatory mechanism in the second intron. Hsc40 is evolutionally conserved. There is over 95% sequence identity between the putative mouse and human hsc40 proteins. However, there is only 60% sequence identity between the hsc40 and hsp40 proteins from either human or mouse cells. Northern blot analysis of various tissues and cells in culture revealed that this gene was expressed under normal conditions, and its expression was further increased after various stress treatments. The expression pattern of the hsc40 gene is very similar to that of the hsc70 gene under both normal and stress conditions, whereas the hsp40 gene exhibited an expression pattern more similar to that of hsp70 genes on the same Northern blots. These results, considered along with the high rate of sequence conservation between the same proteins from different species and high rate of variation between the two different proteins from the same species, strongly suggest that hsc40 and hsp40 may perform different functions and/or have different specificities in the cell.