The species-specific chemical messenger, antheridiogen A(Ce), mediates the differentiation of male gametophytes in the fern Ceratopteris. In order to investigate the biochemical origin of antheridiogen, the effect of the inhibitors, 2'-isopropyl-4'-(trimethylammoniumchloride)-5' -methylphenylpiperidine-1-carboxylate (AMO-1618), 2-chloroethyl trimethylammonium chloride (CCC), and alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) on gametophytic sex expression was determined in C. richardii. Both AMO-1618 and ancymidol blocked the production of male gametophytes in three genetically defined strains of C. richardii that exhibit different sensitivities to antheridiogen. Antheridiogen supplementation overcame inhibition by AMO-1618 and ancymidol, except in one strain (HaC18) that is insensitive to antheridiogen supplementation. These data suggest that the synthesis of Ceratopteris antheridiogen, a taxon that is insensitive to exogenously supplied gibberellins, occurs via a pathway that may include steps in common with gibberellin biosynthesis or involves similar reactions.