cDNAs for three isotypes of inhibitor-2(I-2), I-2 alpha 1, I-2 alpha 2 and I-2 beta were isolated from a rat testis library. I-2 alpha 2 and I-2 beta are new forms. The former is an alternatively spliced form of I-2 alpha 1, encoding a protein with substitution of three amino acids for 14 amino acids of the I-2 alpha 1 protein at the C-terminus. The latter is derived from a different gene and encodes a 126-amino-acid protein having highly conserved regions with the I-2 alpha 1 protein from amino acid positions 22-47 and 111-126. I-2 alpha 2 and I-2 beta are expressed exclusively in the testis, and the expressions of all three forms of I-2 coincide with sperm cell maturation. The half-maximal inhibitory concentration of the GST-I-2 alpha 2 fusion protein on the PP1 gamma 2 catalytic subunit is the same as that of GST-I-2 alpha 1, being 10 nM. However, the half-maximal inhibitory concentration of GST-1-2 beta is 100-fold higher, being 1 microM. GST-I-2 beta showed no competition with GST-I-2 alpha 1. and its biological significance is unknown.