Tributyl phosphate (TBP) and other alkyl phosphates represent a class of persistent organophosphorus compounds of widespread use. Biodegradation of the phosphotriesters is postulated to occur through sequential hydrolytic cleavages via the phosphodiester and monoester intermediates to alcohol and inorganic phosphate (P-i). Immobilized cells of a Citrobacter sp. liberated P-i upon challenge with TBP but the reaction was short-lived. In vitro studies with purified phosphomonoesterase (phosphatase) used P-31 nuclear magnetic resonance to demonstrate P-i transfer onto ethanol (phosphotransferase activity). This suggested that in vivo the onset of a futile phosphohydrolytic and transphosphorylation cycle would limit the extent of phosphate production. A mutant deficient in the transphosphorylating phosphomonoesterase showed an extended release of P-i under challenge with TBP that was not subject to the complete and premature reaction termination that precluded application of the parent strain to possible industrial processes for alkyl phosphate biodegradation.