A general method of confocal laser scanning microscopy was used to demonstrate specific binding of fluorescein-labeled naloxone (FNAL, 10-50 nM) to stably transfected mu opioid receptors on live Chinese hamster ovary cells. Nonspecific binding was visually indistinguishable from autofluorescence in cells with intact cell membranes. Fluorescent labeling of cell perimeters, not present in control nontransfected cells, reversed in transfected cells upon washout of FNAL or following the addition of either unlabeled naloxone (25 microM) or the mu specific antagonist CTOP (1 microM). The addition of the delta and kappa specific agonists DPDPE (1 microM) and U50488 (1 microM), respectively, failed to reverse the labeling. Further evidence of specific binding was obtained from kinetic experiments, where it was observed that only transfected cells showed a time-dependent exponential change in fluorescence that permitted estimation of association and dissociation binding rate constants of (5.8+/-0.5, mean+/-S.E.M.)x10(5) M(-1) s(-1) and (3.3+/-0.6)x10(-3) s(-1), respectively and a kinetically derived dissociation constant of 5.7+/-1.4 nM. These estimates were comparable to those obtained under similar conditions in radioligand binding experiments using [3H]-naloxone.