A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 muM parathion (146 mg 1(-1)) and prevented the accumulation of P-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 muM p-nitrophenol (83 mg 1(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 muM p-nitrophenol (139 mg 1(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.