OBJECTIVE Caffeine has been used as a scaffold for the design of inhibitors of monoamine oxidase (MAO) A and B. Substitution at the C8 position with a variety of moieties yields structures with high MAO inhibition potencies. Although the MAO inhibitory properties of numerous caffeine derivatives have been characterized, the possibility that caffeine inhibits the MAOs has not been investigated in detail. Based on the therapeutic applications and potential adverse effects of MAO inhibition, this study examines the interactions of caffeine with the MAOs. METHODS Employing the recombinant human enzymes, the potencies by which caffeine inhibits the in vitro catalytic activities of the MAOs were recorded and expressed as the IC₅₀ and Ki values. The reversibility of inhibition was determined by measuring the recovery of enzyme activity after dialysis of enzyme-caffeine mixtures. RESULTS Caffeine acts as a MAO inhibitor with Ki values of 0.70 mM and 3.83 mM for the inhibition of MAO-A and MAO-B, respectively. The results show that caffeine binds reversibly and competitively to both MAO enzymes. CONCLUSIONS Although structural modifications of caffeine lead to highly potent MAO inhibitors, caffeine is a weak inhibitor of MAO-A and MAO-B. At plasma concentrations (approximately 1-10 μM) achieved by normal human consumption, the MAO inhibitory potencies of caffeine are unlikely to be of pharmacological relevance in humans. The MAO inhibitory effects of caffeine should however be taken into consideration when using this drug in vitro and in tissue culture experiments where higher doses and concentrations of caffeine are often used.