The concentrations of several antibiotics were measured in the cyst fluid of six adult patients with polycystic kidney disease. Seventy-nine cysts were aspirated at surgery or autopsy. Sixty-one cysts could be categorized as arising from the proximal nephron and 16 from the distal nephron by cyst fluid to serum sodium ratios. Serum, urine, and cyst fluid were simultaneously analyzed for sodium, creatinine, and various antibiotics. Gentamicin, tobramycin, cephapirin, and ticarcillin were either undetectable or present in low concentrations in renal cysts. Cyst fluid antibiotic concentrations did not correlate with cyst volume or creatinine clearance. Cysts of proximal nephron origin had higher antibiotic concentrations than distal cysts. In one patient with normal renal function, inulin was undetectable in renal cysts after a continuous 36-hour i.v. infusion. Para-aminohippurate, however, was detected in the renal cysts of this patient. These data help explain the poor clinical response of infected renal cysts to antibiotic therapy. They also suggest that antibiotics and other solutes may enter cyst fluid across tubular cells in addition to entry by glomerular filtration.