The atrial natriuretic factor (ANF) is comprised of a 126-amino-acid precursor (pro-ANF) and its biologically active fragments. Partially purified pro-ANF and its larger fragments (greater than 10,000 daltons) have been referred to as high-molecular-weight (Mr) ANF, the partially purified smaller fragments (less than 10,000 daltons) as low Mr ANF. In vitro, mild proteolysis of high Mr ANF yielded low Mr ANF and enhanced biological activity. In the rat, pro-ANF was the predominant atrial form; however, low Mr ANF was largely released from isolated perfused hearts, which suggests that conversion of pro-ANF to low Mr ANF occurred immediately before or during secretion. High Mr ANF was also found in the perfusate of isolated rat hearts and in the plasma of rats, which suggests that some pro-ANF was secreted with low Mr ANF. Evidence for extraatrial conversion and activation of pro-ANF comes from two studies. 1) Intra-renal-arterial injection of high Mr ANF had little renal vascular action, whereas its i.v. injection caused renal vascular dilation, which suggests that the renal vasodilatory action of high Mr ANF became activated during circulation. 2) When high Mr ANF was incubated with rat blood or rat platelets in vitro, its natriuretic activity was converted to low Mr ANF within minutes; the platelet-induced conversion was associated with enhanced activity in relaxing aortic smooth muscle.