TRH stimulates the secretion of PRL by clonal GH3 pituitary cells. The studies of the accompanying paper have shown that the secretory response during the first 30-60 min is biphasic (phase I, 0-3 min; phase II, 5-60 min) and that the phase I response may be mediated through mechanisms involving Ca+2 translocation. In previous studies, it has been shown that TRH treatment rapidly induces the breakdown of inositol phospholipids with accompanying diacylglycerol accumulation. In this paper, we present evidence for a possible role for diacylglycerol as a second messenger which mediates the phase II response to TRH. A role for lipid-dependent mechanisms in regulating PRL secretion in GH3 cells was supported by the finding that phospholipase C, phorbol esters, melittin, and exogenous diacylglycerols were effective secretagogues in GH3 cells. Secretion promoted by these agents was found to be persistent, as was the phase II response to TRH. For three of the agents examined (TRH, phorbol esters, and phospholipase C), stimulated PRL release was found to be nonadditive, suggesting the presence of some common element in the pathways by which these agents exert their effects. This lipid-linked pathway of activation was distinguished from a cAMP-mediated pathway.