Labeled and unlabeled 1-acetyl-7-deacetylforskolin and forskolin were synthesized by acetylation of 7-deacetylforskolin with labeled and unlabeled acetyl chloride. The binding of 1-acetyl[1-acetyl-11C]-7-deacetylforskolin ([11C]1-acetyl-7-deacetylforskolin) and [7-acetyl-11C]forskolin ([11C]forskolin) to rat brain membranes was studied using filtration assay. The [11C]forskolin binding was decreased with an increasing load of unlabeled forskolin, whereas [11C]1-acetyl-7-deacetyl-forskolin binding was always very low, the level of which agreed with that of the non-specific binding in forskolin. However, binding of [7-acetyl-11C]1,9-dideoxyforskolin, which has been used as a non-specific inactive analog of forskolin, had a higher binding ratio than that of the non-specific binding of forskolin. The binding of [11C]forskolin was not affected by an increased load of cold 1-acetyl-7-deacetylforskolin. Forskolin activated adenylyl cyclase (AC) in cultured human endothelial cells, whereas 1-acetyl-7-deacetylforskolin did not. These data show that the 1-acetyl-7-deacetylforskolin lacks specific binding affinity and the ability to stimulate AC, while it has similar physical properties with forskolin. The compound 1-acetyl-7-deacetylforskolin would be a suitable "non-specific inactive analog" of forskolin with which to estimate its specific high-affinity binding capacity and to validate forskolin-specific AC stimulation in vitro.