1. A method of extracting and partially purifying a smooth muscle inhibitory factor from the bovine retractor penis is described. This consists of extraction in methanol followed by adsorption on an anion exchange resin, elution from the resin with 500 mM-sodium chloride solution and, if necessary, removal of adenine nucleotides by adsorption on alumina. 2. The inhibitory factor exists in a stable pharmacologically inactive form and an unstable pharmacologically active form. Conversion to the active form is by a brief exposure to acid at pH 2.0. 3. The inhibitory factor is insoluble in ether or acetone but soluble in methanol. Anhydrous methanol, however, irreversibly destroys pharmacological activity especially if the inhibitory factor is in the active form. This effect of methanol is prevented by the presence of 20-30-% water. 4. The inhibitory factor binds to an anion exchange resin but not to a cation exchange resin. It can be eluted from the resin by 500 mM-sodium chloride solution. 5. The molecular weight of the inhibitory factor, as judged by the ability to pass ultrafiltration membranes, is about 500. 6. Inhibitory activity is unaffected by the proteases trypsin, subtilisin or pepsin or by leucine aminopeptidase, pyroglutamate aminopeptidase or carboxypeptidase. The inhibitory effect of the extract and the inhibitory response to stimulation of the non-adrenergic, non-cholinergic nerves are also unaffected by the protease inhibitor, aprotinin. The active material, therefore, is unlikely to be a peptide. 7. Inhibitory activity is abolished by exposure of the extracts to periodic acid or sodium periodate. Acetic anhydride in pyridine also abolishes activity but the vehicle pyridine is also effective. 8. Sodium borohydride but not borate abolishes inhibitory activity when added to the acid-activated material at pH 2.0 but has no effect or may even potentiate activity if added to the stable inactive form at pH 9.0. When added to the acid-activated but neutralized material at pH 6.8 it usually abolishes inhibitory activity but occasionally has no effect. 9. These results suggest the smooth muscle inhibitory factor in these extracts is potent and probably novel. It does not appear to be a peptide or a lipid but may contain a carbohydrate as part of the molecule. Its possible physiological role is discussed.