1. We have shown earlier that pilocarpine strongly inhibits mouse and human liver coumarin 7-hydroxylase activity of CYP 2A and pentoxyresorufin O-deethylase activity of CYP 2B in vitro. Since pilocarpine, like coumarin, contains a lactone structure we have studied in more detail its inhibitory potency on mouse and human liver coumarin 7-hydroxylation. 2. Pilocarpine was a competitive inhibitor of coumarin 7-hydroxylase in vitro both in mouse and human liver microsomes although it was not a substrate for CYP 2A5. Ki values were similar, 0.52 +/- 0.22 microM in mice and 1.21 +/- 0.51 microM in human liver microsomes. 3. Pilocarpine induced a type II difference spectrum in mouse, human and recombinant CYP 2A5 yeast cell microsomes, with Ka values of 3.7 +/- 1.6, 1.6 +/- 1.1 and 1.5 +/- 0.1 microM, respectively. 4. Increase in pH of the incubation medium from pH 6 to 7.5 increased the potency of inhibition of coumarin 7-hydroxylation by pilocarpine. 5. Superimposition of pilocarpine and coumarin in such a way that their carbonyls, ring oxygens and the H-7' of coumarin and N-3 of pilocarpine overlap yielded a common molecular volume of 82%. 6. The results indicate that pilocarpine is a competitive inhibitor and has a high affinity for mouse CYP 2A5 and human CYP 2A6. In addition the immunotype nitrogen of pilocarpine is coordinated towards the haem iron in these P450s.