Lupanine 17-hydroxylase, the first enzyme in the pathway for bacterial degradation of the alkaloid, lupanine, was purified from a Pseudomonas sp. The enzyme acts by initial dehydrogenation of the substrate, and cytochrome c was used as electron acceptor in assays. It had an Mr of 66,000 by ultracentrifuge studies and 74,000 by gel filtration. The visible absorption spectrum was that of a cytochrome c, and a stoicheiometry of one haem group per molecule of enzyme was calculated. SDS/PAGE gave a single band of Mr 72,000 containing the haem group. The enzyme also contained pyrroloquinoline quinone (PQQ), which could be removed by isoelectric focusing. The apoenzyme was reconstituted to full activity with addition of PQQ, and a stoicheiometry of one molecule of PQQ per molecule of enzyme was calculated. Steady-state kinetics gave values of 3.6 microM for the Km for lupanine, 21.3 microM for the Km for cytochrome c and 217 s-1 for the Kcat.