1. Furafylline inhibition of 1A2-related activity in human liver microsomal systems was characterized. This inhibition was time and NADPH dependent. The kinetic constants were measured in human liver microsomes; a Ki of inactivation of 3 microM with a maximum rate constant of 0.27 min-1 were determined. 2. This inactivation process was retarded by the presence of a 1A2 substrate and after complete inhibition was achieved, 1A2 activity could be restored by the addition of fresh microsomes to the incubation mixture. These results are consistent with furafylline being a suicide substrate for 1A2. 3. Preincubating microsomes for 10 min with 10 microM furafylline in the presence of NADPH, prior to the initiation of the reaction by the addition of substrate, caused marked inhibition of 1A2 activity. This protocol was tested for specificity against 10 human P450 activities. The activities associated with 1A1, 2A6, 2B6, 2C9(/8), 2C19, 2D6, 2E1, 3A4(/5) and A were not significantly inhibited. 4. Using these conditions furafylline can be diagnostic of 1A2 involvement in a P450-dependent oxidative reaction.