The triiodinated angiographic contrast medium, iothalamate, has (usually labelled 125I) been used extensively as a marker for glomerular filtration. We have studied the renal handling of 125I iothalamate (IOT) in vivo and in vitro in several species. In renal cortical slices from chicken, rabbit, rat, and monkey, the tissue-to-medium ratio of IOT was twice that of 51Cr-EDTA (EDTA) at 37 degrees C; a difference that was abolished at 0 degree C and markedly reduced by added o-iodohippurate or iodipamide. In five chickens the steady-state renal clearance of IOT (CIOT) was twice (P less than 0.05) that of EDTA (CEDTA) or 3H inulin (C1); a difference that was abolished by administration of 100 mg/kg/hr of novobiocin, an organic anion transport inhibitor. CEDTA was similar to C1 before as well as after transport inhibition. Utilizing the Sperber technique the mean apparent tubular excretion fraction (ATEF) of IOT was 8%, while that of EDTA was 1% (P less than 0.01; N = 10). After novobiocin coinfusion (new steady-state) ATEFIOT was significantly reduced (P less than 0.01) and not different from that of EDTA (-1%). In the same animals the total urinary recovery of IOT was 84 and 57% (P less than 0.01) before and after novobiocin, respectively, while corresponding values for EDTA was unchanged by the inhibitor. In seven rats the renal extraction of IOT was reduced from 29 to 17% (P less than 0.05) by coinfusion of probenecid (5 mg/kg/hr). Corresponding extractions were 82 to 34% (P less than 0.005) and 22% (unchanged) for PAH and EDTA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)