Concentrations of rifampin and trimethoprim, administered in the ratio 3.75:1 were measured in renal hilar lymph, renal interstitial fluid, prostatic interstitial fluid and prostatic secretion in dogs during constant infusion of the antibiotic combination. The lymph was obtained by direct cannulation of the lymphatics and the interstitial fluids from small plastic tissue chambers implanted 4 weeks before the experiments. Unilateral hydronephrosis was produced by ureteral ligation in dogs with tissue chambers in both kidneys to compare concentrations of the drug combination in renal interstitial fluid and urine from normal and hydronephrotic kidneys. Rifampin produced lower concentrations in both prostatic interstitial fluid and prostatic secretion than in plasma, while trimethoprim concentrations were much higher in prostatic secretion and prostatic interstitial fluid than in plasma. Renal lymph/plasma ratios and renal interstitial fluid/plasma ratios were significantly higher for trimethoprim than for rifampin, but only the lymph concentrations of trimethoprim exceeded the corresponding plasma concentrations. High urine concentrations were achieved for both drugs except in pelvises of hydronephrotic kidneys, where virtually no drug activity could be detected. Renal interstitial fluid concentrations of both drugs were similar in hydronephrotic and normal kidneys. The ratios between rifampin and trimethoprim concentrations varied considerably among plasma, various tissues and tissue fluids.