Using immunohistochemistry and in situ hybridization, it has been previously shown that gonadotropes and folliculo-stellate cells in the rat anterior pituitary gland express nitric oxide (NO) synthase (NOS), and that NOS expression is increased by gonadectomy. Using the indirect immunofluorescence technique in conjunction with antibodies raised to conjugated cGMP, we have attempted to establish the target cells for NO in the anterior pituitary and to define the mediator of NO regulation. After incubation of pituitary slices with several NO donors, numerous endocrine cells, but no folliculo-stellate cells, expressed cGMP. Most of these cells stained for LH, that is they were gonadotropes. However, there were apparently cGMP-positive, LH-negative and LH-positive, cGMP-negative endocrine cells. The increase in cGMP could be virtually completely blocked by a guanylyl cyclase inhibitor. cGMP was not expressed in corticotropes, but cGMP-positive cells often contained NOS-like immunostaining. Incubation with GnRH did not result in detectable levels of cGMP. However, when castrated rats were pretreated with a potent longlasting GnRH antagonist, antide, the castration-induced increase in NOS was completely blocked. This suggests that GnRH is involved in the in vivo upregulation of NOS after castration, but that GnRH cannot induce cGMP accumulation in normal pituitary slices in vitro. Taken together, the present results give further evidence for a role of NO in the control of, in particular, LH secretion from the anterior pituitary gland in the rat.