Milt from Pacific herring (Clupea harengus pallasi) was extracted and purified on reversed-phase high-performance liquid chromatography. The fractions were radioimmunoassayed for the following free and conjugated steroids: cortisol; 17α,20β-dihydroxy-4-pregnen-3-one; 17α,20α-dihydroxy-4-pregnen-3-one; 17α,21-dihydroxy-4-pregnene-3,20-dione; 17α,20β,21-trihydroxy-4-pregnen-3-one; 17α-hydroxy-4-pregnene-3,20-dione; 3α,17α,21-trihydroxy-5β-pregnan-20-one; testosterone; 11-ketotestosterone. Fractions containing significant amounts of immunoreactive material were subjected to further purification on thin-layer chromatography. Substantial amounts of cortisol (271 ng∙g−1 of milt) and 17α,20β-dihydroxy-4-pregnen- 3-one (ca. 38 ng∙g−1 of milt) were found in the free fractions of one of the extracts. Substantial amounts of cortisol (229 ng∙g−1 of milt), 17α,20β-dihydroxy-4-pregnen-3-one (25.7 ng∙g−1 of milt), and 3a, 17α,21-trihydroxy-5β-pregnan- 20-one (13 ng∙g−1 of milt) were found in the conjugated fractions of both extracts. Levels of the other steroids (free and conjugated) ranged from undetectable (<0.1) to 5.9 ng∙g−1 of milt. The possible reasons for the differences in free steroid levels between the extracts, and the potential role of the steroids as pheromones, are discussed.