Pseudomonas aeruginosa 579 biofilms formed on dialysis membranes retarded piperacillin diffusion. Treatment of biofilms with 5.0 mM CaCl2.2H2O prevented diffusion. Biofilms permitted equilibration of [14C]glucose. Thin-layer chromatography of fluids distal to untreated (viable and nonviable) and viable Ca(2+)-treated P. aeruginosa 579 biofilms and fluids distal to a viable P. aeruginosa mutant noninducible for the expression of beta-lactamase did not detect piperacillinoic acid.