The renal transport of 125I-diatrizoate was examined in three species, dog, rabbit, and rat. In the rabbit this organic acid behaved like many such compounds, i.e. it was secreted by the proximal tubular organic anion process as judged by stop-flow analysis. The proximal tubular peak was abolished by probenecid. In vitro experiments with renal cortex slices gave results entirely consistent with the in vivo studies. Diatrizoate uptake by the slices was an energy-dependent, saturable process that was blocked by several organic acids known to be accumulated by the organic acid transport system. Rat renal cortex slices showed significant diatrizoate uptake, although of a lesser magnitude than noted with rabbit tissue (rat S/M = 1–2; rabbit S/M = 3–5). Except for the failure of acetate to stimulate diatrizoate uptake, accumulation by the rat slices appeared to be by the organic acid transport process. On the other hand, dog slices failed to accumulate diatrizoate. The process was not stimulated by acetate or any other metabolic substrate nor was it blocked by metabolic inhibitors or various organic acid competitors. No evidence of proximal tubular secretion was noted in the dog using stop-flow analysis; diatrizoate-inulin U/P ratios were approximately 0.9. Furthermore in p-amino-hippurate (PAH) clearance experiments in the dog, administration of diatrizoate failed to alter PAH clearance. These data indicate active renal transport of diatrizoate in certain species, but not in others.