1. (1-Methyl-5-piperazine-1-yl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-(5-methyl-pyridin-2-yl)-amine (UK-469,413) was identified as a lead compound in a new medicinal chemistry programme. UK-469,413 had good physicochemical properties and was slowly metabolized by cytochromes P450 in rat and human liver microsomes. 2. In the rat in vivo the compound was rapidly cleared. Subsequent studies showed that UK-469,413 was rapidly acetylated in rat liver cytosol to an N-acetylpiperazine metabolite that was the major circulating metabolite in rat plasma in vivo. 3. Analogues of UK-469,413 containing the unsubstituted piperazine moiety were rapidly acetylated in rat liver cytosol and had high plasma clearance in the rat in vivo. These compounds were also acetylated in human liver cytosol and the N-acetyl metabolite was a major metabolite formed in incubations with cryopreserved human hepatocytes. 4. Using specific inhibitors, correlation analysis and expressed human N-acetyltransferase (NAT) enzymes the compounds were shown to be substrates of the polymorphically expressed NAT-2 isozyme. 5. Further experiments showed that it was possible to make small structural changes to the piperazine group that retained potency but prevented metabolism by NAT.