To investigate the participation of cyclic nucleotides in LHRH-mediated gonadotropin release, cells from the anterior pituitaries of 15-day-old female rats were fractionated on an albumin gradient by sedimentation at unit gravity. Gonadotrophs and somatotrophs were detected in the fractions by histology and RIA of hormone content, then the cells were pooled into three subpopulations and cultured overnight. The effect of LHRH on hormone release and cyclic nucleotide content was examined by incubation in the presence or absence of the releasing hormone. After 60 min, LHRH (5 nin) had induced a 5- to 6-fold increase in LH release only from the cells in the subpopulation which was enriched in gonadotrophs. At no time did LHRH influence cAMP levels in any of the cells; however, the cGMP content of the cells in the gonadotroph-containing pool rose to twice that in the controls after only 15 min in the presence of LHRH. The increase in cGMP concentration in the cells was at or near maximum by the time of the init ial sampling; however, LH continued to accumulate in the medium over the entire test period. These observations support the view that cGMP is involved in the action of LHRH.