Piclamilast is a type 4 phosphodiesterase (PDE4) inhibitor with equal affinity for the high-affinity rolipram binding site (HARBS) and low-affinity rolipram binding site (LARBS). The binding of [(3)H]piclamilast to preparations of rat brain and peripheral tissue was investigated and compared with that of [(3)H]rolipram. [(3)H]piclamilast binding was high-affinity, saturable, reversible, and partially Mg(2+)-dependent. Binding was detected both to membrane and soluble fractions, with K(d) values of 3.1 and 4.5 nM, respectively. The B(max) values for [(3)H]piclamilast were about 1.5-fold greater than that of [(3)H]rolipram binding, suggesting that [(3)H]piclamilast, but not [(3)H]rolipram, binds to LARBS as well as the HARBS. The HARBS was present in all the brain regions examined, but not in peripheral tissues. All PDE4 inhibitors tested were potent competitors for [(3)H]piclamilast binding; the competition curves for rolipram, desmethylpiclamilast, ICI 63,197, and Ro 20-1724 were better described by a two-site model, while the competition curves for piclamilast, cilomilast, roflumilast, and CDP 840 were adequately described by a one-site model. Inhibitors of other PDE families were much less potent. The inhibition of [(3)H]piclamilast was further tested in the presence of 1 microM rolipram to isolate the LARBS. Under this condition, the competition curves for all the inhibitors were adequately described by a one-site model, with K(i) values close to that for the LARBS. The results indicated that [(3)H]piclamilast is a useful tool to directly study inhibitor interaction with the HARBS and the LARBS in rat brain.