The objective was to test antibiotic activity against Staphylococcus epidermidis and Pseudomonas aeruginosa in fresh dianeal fluid and spent dialysate from patients undergoing chronic ambulatory peritoneal dialysis. MICs and MBCs were measured and compared with those in standard broth. For S. epidermidis, there was some reduction in cefazolin and vancomycin activity in dialysis fluids. For P. aeruginosa, ciprofloxacin and tobramycin MICs increased significantly, and all isolates were tolerant to ceftazidime and piperacillin in dialysis fluids. Dialysis fluids can significantly impair antibiotic activity. The clinical implications warrant further study of antibiotic pharmacodynamics in the treatment of peritonitis.