The isolation of a rat hsp 70-related gene which is specifically and highly expressed in testis is described together with the complete nucleotide sequence of the transcription unit (2947 bp), 5' flanking (about 1 kbp) and 3' flanking (about 0.3 kbp) regions. The sequence analysis and nuclease S1 mapping revealed that the isolated gene (referred to as the hst70 gene) represents a novel, distinct member of the hsp70 multigene family. Its transcription unit lacks introns and a single open reading frame encodes a protein of 69.5 kDa. The predicted amino acid sequence of this protein is highly similar (only four out of 633 amino acids are different) to that encoded by the mouse testis-specific hsp70.2 gene (Zakeri, Z.F., Wolgemuth, D.J. and Hunt, C.R. (1988) Mol. Cell. Biol. 8, 2925-2932). The functional significance of multiple potentially regulatory sequences (e.g. TATA-boxes, heat-shock element and estrogen receptor binding site) present in the 5' flanking region of the rat hst70 gene is discussed.