The regulation of the atrial natriuretic factor (ANF) receptor system in cultured rat vascular smooth muscle cells (RVSMC) was examined following long term pretreatment of these cells with rANF99-126 or with any one of a series of truncated and ring-deleted analogs. The latter analogs are reported to bind selectively the ANF-C or clearance receptor. Initial competition binding studies revealed that all analogs examined showed comparable apparent receptor binding affinities (Ki values did not differ by more than 10-fold). In contrast, the extent of interaction of the ANF analogs with the receptor pool coupled to particulate guanylate cyclase (the ANF-B receptor) was much more variable, with some ligands failing to stimulate cGMP production or particulate guanylate cyclase over the concentrations tested. Pretreatment of cells for 24 h with rANF99-126 or any of the truncated analogs that interact with the ANF-B receptor caused a dose- and time-dependent decrease in the number of ANF binding sites (99% of which are uncoupled in RVSMC) without any change in affinity. Examination of the binding activity following pretreatment of the cells with ANF suggested that the observed reduction in 125I-rANF99-126 binding capacity was not because of the retention of the peptide on its receptor. Furthermore, this down-regulation was associated with desensitization of particulate guanylate cyclase resulting in a decreased responsiveness of intracellular cGMP accumulation to ANF. In contrast, however, analogs selective for the ANF-C receptor pool failed to cause down-regulation or desensitization. These findings suggest that ANF-C receptors in RVSMC are not independently down-regulated by selective ligands but that nonselective analogs that down-regulate and desensitize the ANF-B receptor system can by some cooperative mechanism reduce the size of the predominant ANF-C receptor pool in these cells.