The binding of [125I]LSD in rat brain was characterized and localized by quantitative autoradiography. Frozen 32 microns thick brain sections were labeled in vitro with [125I]LSD and exposed for 24 h against LKB Ultrofilm to generate autoradiograms. Nonspecific binding was defined as the labelling in the presence of 1 microM D-LSD. Scatchard analysis by densitometry indicated that the binding of [125I]LSD was saturable, with a Kd of 0.2 nM. In agreement with the results of Hartig and co-workers [5,6], [125I]LSD showed a high affinity for 5-HT2 serotonin receptors with some overlap with D-2 dopamine receptors. High concentrations of [125I]LSD sites were observed in layer IV of the cerebral cortex, caudate-putamen, claustrum, olfactory tubercle, nucleus accumbens, ependyma, mammillary nucleus and inferior olive. Co-incubation of sections with sulpiride to block binding to D-2 receptors resulted in a uniform 20-30% reduction in the amount of specific [125I]LSD binding, with no qualitative difference in the pattern of labeling. However, co-incubation with ketanserin to block 5-HT2 receptors resulted in a pattern of binding that was similar to previous descriptions of the location of D-2 receptors, with high levels of residual binding in caudate-putamen, olfactory tubercle, nucleus accumbens and inferior olive. Our results indicate that [125I]LSD is a suitable ligand for quantitative autoradiography of both 5-HT2 and D-2 receptors, and that there is a strong anatomical correspondence between these receptor subtypes, perhaps implying a functional interaction.