We have determined the effect of naltrexone, naloxone, [D-Ala2,D-Leu5]enkephalin (DADLE), and morphine on the mu-S196A opioid receptor knock-in and mu-opioid receptor knockout mouse vas deferens preparations. The antagonists, naltrexone and naloxone, exhibited agonist activity and possessed IC50 values that were 14- and 37-fold greater than morphine on the S196A preparation. Morphine was found to be threefold more potent at S196A relative to wild-type mu-opioid receptor. The mouse vas deferens data suggest that S196 in transmembrane helix 4 of the mu-opioid receptor modulates efficacy. It is proposed that this may be due to decreased dimerization of the receptor. Identical IC50 values of DADLE obtained on the wild-type, S196A knock-in, and mu-opioid receptor knockout preparations support the absence of mu-delta heterodimers in the mouse vas deferens.