We describe the use of fetal rat lung fibroblasts (RFL-6 cells) as a bioassay for endothelium-derived relaxing factor or nitric oxide (EDRF/NO). In these cells grown to confluency in 6-well plates, authentic NO, at concentrations as low as 2 nM, or EDRF/NO, basally released from as few as 1-2 × 106 endothelial cells, causes accumulation of guanosine 3′,5′-cyclic monophosphate. If cells are grown in 48-well plates (well area 1 10 that of 6-well plates) this gives a detection limit of 100-200 fmol NO and the possibility of detecting the basal EDRF/NO release from 1-2 × 105 endothelial cells. Thus, this method is more sensitive than any other currently available. In addition, RFL-6 cells may be used to detect the activity of EDRF/NO synthase in cell homogenates and column fractions during purification, making them an invaluable aid in purification and characterization of EDRF/NO synthase from brain and neuronal cells. © 1992.