Microchlorination of 1,4,9[3H]dibenzofuran gave several polychlorinated dibenzofuran (PCDF) products and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-[3H]pentachlorodibenzofuran (PeCDF), and 1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) of high specific activity (57, 34, and 32.5 Ci/mmol, respectively) were purified by preparative high-pressure liquid chromatography. These compounds were investigated as radioligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor protein. Like 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD), the radiolabeled PCDF congeners exhibited saturable binding with the receptor protein and sucrose density gradient analysis of the radiolabeled ligand-receptor complexes gave specific binding peaks with comparable sedimentation profiles. The rank order of radioligand binding affinities (Kd values) was 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,6,7,8-HCDF greater than 1,2,3,7,8-PeCDF and the maximum difference in Kd values for the four radioligands was less than 13-fold (0.44-5.9 nM). The interactions of the PCDF radioligands with the cytosolic receptor all exhibited saturable binding curves and linear Scatchard plots and the slopes of their Hill plots were in the range 1.0-1.1, thus indicating that cooperativity was not a factor in these binding interactions. The relative stabilities and dissociation kinetics of the radioligand-receptor complexes were highly dependent on the structure of the radioligand. The dissociation curves of the 2,3,7,8-[3H]TCDD and PCDF receptor complexes were biphasic and this suggests that there may be a temporal shift in ligand binding affinities. However, the rates of dissociation did not correlate with the rank order of ligand binding affinities. The stabilities of the radioligand-receptor complexes were also dependent on the structures of the radioligands; for example, the 2,3,7,8-[3H]TCDD-receptor complex degraded more rapidly than the PCDF-receptor complex and these relative stabilities were clearly not related to the Kd values or the relative in vivo or in vitro biologic potencies of these halogenated aryl hydrocarbons.