In response to a recent report (Lewis, A.S., Murphy, L., Mcalla, C., Fleary, M., and Purcell, S. (1984) J. Biol. Chem. 259, 12-15) that folic acid was a potent inactivator of xanthine oxidase, the details of this apparent inactivation were studied. In confirmation, we also found that commercially available folic acid produced a time-dependent progressive inhibition (apparent inactivation) of xanthine oxidase. A plot of the pseudo-first order rate constant of the decay of enzyme activity versus the concentration of folic acid resulted in a straight line. This indicated that the progressive inhibition was caused by a slow second order combination of an inhibitor with the enzyme. The second order rate constant for this association (slope of replot) was 5.7 X 10(3) M-1 S-1. The slowness of this constant together with the observation that complete inactivation did not occur suggested that the progressive inhibition might be due to the slow binding of a high affinity contaminant. This was corroborated by the finding that the association constant was decreased to 1.6 X 10(2) M-1 S-1 after partially purifying the folic acid. The compound most likely to be producing this inhibition is pterin aldehyde (2-NH2-4-OH-pteridine-6-aldehyde), a photolytic breakdown product of folic acid. Pterin aldehyde was found to be a progressive inhibitor of xanthine oxidase with an association constant of 2.2 X 10(5) M-1 S-1. When the apparent association constants of commercial and purified folic acid were adjusted to reflect the pterin aldehyde content (3.6% and 0.2%, respectively), they became similar to the association constant of pterin aldehyde. Thus, it seems that the apparent inactivation of xanthine oxidase by folic acid was caused by the slow binding of contaminating pterin aldehyde.