1. Pseudomonas N.C.I.B. 9872 grown on cyclopentanol as carbon source oxidized it at a rate of 228mul of O(2)/h per mg dry wt. and the overall consumption of 5.9mumol of O(2)/mumol of substrate. Cyclopentanone was oxidized at a similar rate with the overall consumption of 5.2mumol of O(2)mumol of substrate. Cells grown with sodium acetate as sole source of carbon were incapable of significant immediate oxidation of these two substrates. 2. Disrupted cells catalysed the oxidation of cyclopentanol to cyclopentanone by the action of an NAD(+)-linked dehydrogenase with an alkaline pH optimum. 3. A cyclopentanolinduced cyclopentanone oxygenase (specific activity 0.11mumol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mumol of NADPH and 0.9mumol of O(2) in the presence of 1mumol of cyclopentanone. NADPH oxidation did not occur under anaerobic conditions. The only detectable reaction product with 100000g supernatant was 5-hydroxyvalerate. 4. Extracts of cyclopentanol-grown cells contained a lactone hydrolase (specific activity 7.0mumol hydrolysed/min per mg of protein) that converted 5-valerolactone into 5-hydroxyvalerate. 5. Cyclopentanone oxygenase fractions obtained from a DEAE-cellulose column were almost devoid of 5-valerolactone hydrolase and catalysed the formation of 5-valerolactone in high yield from cyclopentanone in the presence of NADPH. 6. Incubation of 5-hydroxyvalerate with the 100000g supernatant, NAD(+) and NADP(+) under aerobic conditions resulted in the consumption of O(2) and the conversion of 5-hydroxyvalerate into glutarate. 7. The high activity of isocitrate lyase in cyclopentanol-grown cells suggests that the further oxidation of glutarate proceeds through as yet uncharacterized reactions to acetyl-CoA. 8. The reaction sequence for the oxidation of cyclopentanol by Pseudomonas N.C.I.B. 9872 is: cyclopentanol --> cyclopentanone --> 5-valerolactone --> 5-hydroxyvalerate --> glutarate --> --> acetyl-CoA.