Gonadotropic hormones (GTHs) were purified from Atlantic croaker (Micropogonias undulatus) pituitary glands collected in December, when the gonads were regressed. GTH was identified during the purification by an in vitro ovarian steroidogenesis bioassay and by radioimmunoassay using an Atlantic croaker maturational GTH antiserum. Two gonadotropic fractions were separated by anion-exchange chromatography of gel-filtered pituitary extracts. Reverse-phase HPLC followed by n-terminal amino acid sequencing of the GTH subunits revealed that a common alpha-subunit was present in both fractions. However, the two gonadotropic fractions contained different forms of the beta-subunit which differed both in their n-terminal amino acid sequences and in their amino acid compositions. The differences in the amino acid compositions between the two croaker GTH beta-subunits were similar to those previously reported between salmon GTH I and II beta-subunits (Kawauchi et al., 1989, Fish Physiol. Biochem. 7, 29-38). The n-terminal sequence of one of the croaker beta-subunits (beta-II) resembled that of the salmon GTH II beta-subunit. The other subunit (beta-I) had only a low degree of homology in its n-terminal sequence with the salmon GTH I beta-subunit but had a similar amino acid composition. In addition, the n-terminal end of beta-I had 40% sequence identity with the GTH I beta-subunit of bonito (Kawauchi et al., 1991, In "Proceedings of the Fourth International Symposium on Reproductive Physiology of Fish (A. P. Scott, J. P. Sumpter, D. E. Kime, and M. S. Rolfe, Eds.), pp. 19-21. Univ. of East Anglia, Norwich, UK). Moreover, the beta-I-subunits from all three species had cysteine residues at positions 4 and 8. These results indicate that a dual gonadotropin system exists in the Atlantic croaker as in the salmon and bonito and support the concept that such a system is ubiquitous in teleosts.