The existence of the peripheral-type benzodiazepine receptor (PBR) in vascular smooth muscle has been demonstrated in this laboratory. The present study utilized the photoaffinity ligand [3H]PK14105 to identify the protein subunit in rat aortic and other smooth muscle types to which high affinity ligands for the PBR bind. [3H]PK14105 bound to mitochondrial fractions isolated from rat aortic smooth muscle with high affinity (Kd = 7.0 +/- 0.5 nM) and high density (Bmax = 10.1 +/- 1.5 pmol/mg protein). The rank order of potency of a series of PBR ligands displacing the binding was PK11195 approximately equal to Ro5-4864 greater than protoporphyrin IX greater than flunitrazepam greater than diazepam much greater than clonazepam. [3H]PK14105 bound with comparable affinity and density to mitochondria isolated from rat myometrium and gastric smooth muscle as well. With ultraviolet irradiation, [3H]PK14105 specifically labeled a single protein of approximately 17 kDa in all three smooth muscle types examined. This protein was identical in size to that identified by [3H]PK14105 in rat adrenal gland. In adrenal gland an additional, minor protein of approximately 43 kDa was also specifically labeled by [3H]PK14105. Utilizing a probe designed from the known nucleotide sequence of the PBR in rat adrenal gland, an mRNA transcript of approximately 0.8 kilobases in size was identified in rat aortic smooth muscle by Northern blot analysis. These data indicate that a protein subunit of approximately 17 kDa comprises, at least in part, the PBR not only in vascular smooth muscle, but also in other smooth muscle types and adrenal gland as well.