Both [3H]U69,593 alone and [3H]ethylketocyclazocine (EKC) in the presence of mu and delta blockers ([D-Ala2,MePhe4,Glyol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE)) label kappa receptors in the guinea pig cerebellum. Dynorphin A(1-17) and nor-binaltorphimine (nor-BNI) potently competed the binding of both radioligands with Hill coefficients of approximately unity, strongly supporting a kappa classification of binding. However, saturation studies revealed that the Bmax for [3H]EKC binding was 45% greater than that for [3H]U69593, suggesting that [3H]EKC might be labeling more than one site. Although nonlinear regression analysis of dynorphin A(1-17) and nor-BNI competition of [3H]EKC binding best fit the curves with a one site model, competitions by dynorphin B, dynorphin A(1-9) and alpha-neoendorphin revealed Hill coefficients less than unity and were best fit to a two site model. Kinetic analysis also supported [3H]EKC binding heterogeneity. Together, these studies imply that under these conditions [3H]EKC labels more than one site in the guinea pig cerebellum. The sensitivity of all specific [3H]EKC binding to the selective kappa ligands dynorphin A(1-17) and nor-BNI indicates that both component are kappa while the differing sensitivities of dynorphin B, alpha-neoendorphin and dynorphin A(1-9) for these components support our previous hypothesis of kappa 1a and kappa 1b binding subtypes.