D-Ala2,Leu5,Cys6-enkephalin (DALCE) is a synthetic enkephalin analog which contains a reduced sulfhydryl group. It exhibited moderate delta selectivity (mu/delta IC50 ratio 13), but was not as selective as the disulfide-containing peptide, D-Pen2,5-enkephalin (DPDPE) (mu/delta ratio 1121). However, unlike other delta-selective peptides, DALCE exhibited a markedly slowed dissociation from receptors after pretreatment of membranes with micromolar concentrations. Pretreatment of membranes with 10 uM DALCE, followed by extensive washing, produced an 85-90% loss of 3H-DPDPE binding sites. D-Ala2,D-Leu5-enkephalin (DADLE), D-Ser2,Leu5,Thr6-enkephalin (DSTLE) and DPDPE produced losses of 59%, 70%, and 19%, respectively. The effect of DALCE was not reversed by a 60 min post-incubation in buffer containing 250 mM NaCl + 100 uM GMPPNP, a condition which produced nearly complete reversal of loss of sites by DADLE and DSTLE. DPDPE could be dissociated merely by post-incubation in TRIS-buffer alone for 15 min. The order for ease of dissociation after preincubation was DPDPE much greater than DADLE greater than DSTLE much greater than DALCE. The effect of DALCE was selective for delta sites, although higher concentrations of DALCE produced loss of mu sites. DALCE pretreatment had no effect on recovery of kappa sites. These results indicate that DALCE binds essentially irreversibly to delta receptors.