Direct screening of preselected compounds in a rat primary pituitary cell culture assay, followed by chemical modification of selected pharmacophores led to the identification of a novel non-peptidyl class of GH secretagogues (substituted benzolactams). The prototype compound of this class, L-692,429, stimulated GH release from rat primary pituitary cells in a time- and dose-dependent manner with an EC50 value of 60 nM. Under the same conditions, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GH-releasing peptide, GHRP-6) and GH-releasing factor (GRF) had EC50 values of 10-8 and 5 × 10-10M, respectively. L-692,428, the S-enantiomer, of L-692,429, was inactive at a concentration as high as 2 μM. GH release induced by L-692,429 was inhibited by somatostatin as well as by GHRP-6 and substance P antagonists but not by GRF or opiate antagonists. L-692,400, which is structurally related to L-692,429 but biologically inactive, inhibited GH response not only to L-692,429 but also GHRP-6. Like GHRP-6, L-692,429 alone had no effect on intracellular cAMP levels; however, it synergized with GRF to further increase both the accumulation of cAMP and the release of GH. Maximal effects of L-692,429 and GHRP-6 on GH release were comparable. Interestingly, when presented together in maximal concentrations, L-692,429 and GHRP-6 did not cause an additional GH release when compared with either secretagogue alone. L-692,429 had a small effect on prolactin release but not adrenocorticotropin. These results demonstrate that L-692,429 is the first non-peptidyl secretagogue which has a direct and specific effect on GH release from rat pituitary cells, and its action appears to be mediated through the same receptor and signalling pathway as GHRP-6.