Detailed characteristics of a new bioassay method for detection and quantification of endothelium-derived relaxing factor (EDRF) are described. Guanosine 3',5'-cyclic monophosphate (cGMP) responses of RFL-6 rat fetal lung fibroblast cells were utilized to estimate the activity of nitric oxide (NO) and EDRF. The conditioned medium from bovine aortic endothelial (BAE) cells cultured in tissue culture plates was quickly transferred to RFL-6 incubations to determine EDRF. In the presence of superoxide dismutase, RFL-6 cells cultured in six-well tissue culture plates exhibited very high sensitivities to both NO and EDRF; e.g., they responded to NO at a concentration as low as 2 nM and the basal release of EDRF from 1-2 X 10(6) BAE cells. Based on the lower detection limit of the radioimmunoassay for cGMP, calculations reveal that 100-200 fmol of NO and the basal EDRF release from 1-2 X 10(5) BAE cells can be detected with RFL-6 cells by choosing smaller culture wells. Thus this method is more sensitive than any other currently available. Furthermore, it may be widely used, since the instrumentation required is presently available in many laboratories. This bioassay technique for EDRF and NO is sensitive, simple, and quite useful for the evaluation of experimental conditions and compounds that regulate EDRF release from various endothelial cells and tissues.