We theorized that H-8, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic nucleotide-dependent protein kinases, might be a useful probe to assess cyclic nucleotide-dependent relaxation of blood vessels. However, working in the rat caudal artery and aorta, we found that neither cyclic AMP- nor cyclic GMP-mediated relaxations were diminished by even large doses of H-8. For example, in the caudal artery, relaxation of a phenylephrine contraction by 8-bromo-cGMP (10<sup>–5</sup> M) was unaffected by H-8: control, 33 ± 6.2%; 10 µ M H-8, 41 ± 12%; 30 µ M H-8, 30 ± 16% (p NS). The amount of relaxation by 8-bromo-cAMP (3 × 10<sup>–4</sup> M) was actually increased by H-8: control, 29 ± 7.6%; 10 µ M H-8, 34 ± 7.4%; 30 µ M H-8, 80 ± 14%. In the rat aorta, H-8 also failed to diminish relaxation induced by 8-bromo-cGMP, or by atriopeptin II or sodium nitroprusside. In both caudal artery and aorta, H-8 of itself caused a dose-dependent suppression of α-adrenergic contraction: for example, in the caudal artery, with 10 or 30 µ M H-8, peak contraction to phenylephrine was reduced to 70 (SEM) ± 12% or 52 ± 7% of control, respectively. The results suggest that the protein kinase inhibitor H-8 is not a useful probe to study cyclic nucleotide-dependent relaxation.