We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase A, that catalyzes the hydrolysis of para-nitrophenylacetate with high affinity (Km approximately 25 microM) and is very sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF). Based on its catalytic properties, isoelectric point, and N-terminal amino acid sequence, hydrolase A corresponds to the pI 6.1 esterase cloned from a rat liver cDNA library by Robbi et al. (Biochem. J. 269, 451-458, 1990). A PMSF-sensitive esterase with high affinity toward para-nitrophenylacetate is also present in testicular microsomes at levels that slightly exceed those in liver microsomes. Antibody against purified hydrolase A recognizes a 57-kDa protein in both liver and testicular microsomes, suggesting that hydrolase A is expressed to a high degree in both tissues. To determine whether the testicular carboxylesterase is identical to hydrolase A, a rat testicular cDNA library was constructed and screened with antibody against hydrolase A. A 709-bp cDNA was isolated from immunopositive clones. Screening the same cDNA library by polymerase chain reaction (PCR) with one primer based on the sequence of the 709-bp cDNA and one primer based on the sequence of the adjoining lambda gt11 arm yielded a 1.1-kb cDNA that overlapped with the 709 bp-sequence. Together these two cDNA fragments spanned a 1792-bp sequence with an opening reading frame encoding 518 amino acids, which corresponds to approximately 95% of the C-terminal sequence of the liver pI 6.1 esterase (i.e., hydrolase A). Except for four nucleotide differences at positions 479, 855, 1335, and 1350, the sequence of the testicular cDNA was identical to the cDNA sequence of the liver pI 6.1 esterase reported by Robbi et al. None these changes results in an amino acid substitution. However, these four base substitutions were not observed when a cDNA encoding hydrolase A was isolated from a rat liver cDNA library by PCR. These results establish that the same carboxylesterase, namely, hydrolase A, is expressed in rat liver and testis. The levels of mRNA for hydrolase A in various rat tissues was estimated from Northern blots probed with the 709-bp cDNA isolated from the rat testicular cDNA library. A approximately 2-kb mRNA for hydrolase A was detected in liver, testis, lung, and prostate, which confirms the tissue distribution of hydrolase A based on catalytic activity and Western immunoblotting.(ABSTRACT TRUNCATED AT 400 WORDS)