Two eicosanoid fractions were isolated by thin-layer chromatography from the homogenates of ten species of barnacles, in an investigation on the in vitro production in barnacle tissues of eicosanoids with egg-hatching activity. The fractions consisted of a monohydroxy fatty acid fraction (MHFA) and a trihydroxy fatty acid (THFA) fraction. Both fractions caused egg hatching and the release of larvae from isolated barnacle egg masses. In an attempt to determine the natural eicosanoid responsible for egg hatching in barnacles in vivo, eicosanoids were extracted from seawater in which Balanus balanoides or B. hameri were hatching and liberating larvae from the mantle cavity. After removal of the newly released larvae from the seawater only a THFA fraction was found that caused egg hatching. The results support the conclusion that the natural eicosanoid with hatching activity (hatching factor) is not an MHFA. It is present in the THFA fraction and is secreted into the mantle cavity. After causing egg hatching any excess is expelled with the larvae into the sea.