The binding sites labelled by 3H-dihydromorphine, 3H-ethylketocyclazocine and 3H-D-Ala2-L-Leu5-enkephalin in mouse brain membranes were characterized in cross-competition studies. The data was evaluated by simultaneous non-linear least-squares regression analysis with the "Ligand" program (Munson & Rodbard 1980), that had to be upgraded to handle more than three binding sites. By statistical analysis four different binding sites were identified. Three of the sites probably correspond to the pharmacologically well characterized mu, kappa and delta-opioid receptors, respectively, and their binding capacities relate as 1:1.5:2.5. Classification of the fourth site is more problematic. Using 3H-ethylketocyclazocine it had higher capacity than the others and bound ethylketocyclazocine with a relatively high affinity (Kd = 10 nM), dihydromorphine with a very low affinity (Kd greater than 10(-5)M) and showed no binding of D-Ala2-L-Leu5-enkephalin. In displacement studies, N-allylnorcyclazocine (SKF 10,047), though unselective, bound with highest affinity to the mu and the fourth site. Since naloxone did not bind to this fourth site, it can not be termed an opioid site in a strict sense, but it might have some relevance in view of non-naloxone-reversible effects reported for some opioids.