We report the cloning and sequencing of 5'-terminal region of a beta form of rat ECE-1 cDNA which is different only in its N-terminal amino-acid sequence to the cDNA we have cloned previously (alpha form [K. Shimada et al. (1994) J. Biol. Chem. 269, 18275-18278]). No significant difference was found in the specific activity and substrate specificity between the two isoforms. The expression level of ECE-1 alpha mRNA was higher than that of ECE-1 beta in various rat cells and tissues, suggesting that the physiologically important isoform is ECE-1 alpha. The present findings verified the presence of two forms of ECE-1 over many species, which are created probably through alternative splicing.