Residual renal function (RRF) may contribute significantly to the total dialysis prescription. Conventional quantitation of RRF in hemodialysis (HD) patients is measured by urea clearance and requires a 24-hour urine collection which is often difficult to perform and inaccurate. The renal clearance of iohexol was evaluated as an alternative method for RRF assessment (iohexol-derived RRF) in hemodialysis patients. An intravenous bolus of iohexol (12 ml; 300 mg iodine/ml) was administered to 42 hemodialysis patients following routine HD. A single blood sample was obtained approximately 44 hours later (pre-HD) to determine the plasma clearance of iohexol using x-ray fluorescence methods. Total body clearance of iohexol (C(NRio)) and non-renal clearance of iohexol (C(NRio)) 2.87 ± 0.3 ml/min (mean ± SEM) were used to calculate iohexol-derived RRF (C(TBio) - C(NRio)) Iohexol-derived RRF determinations were then compared to urea clearance-derived RRF measurements. The RRF contribution to the dialysis prescription was also calculated utilizing iohexol-derived RRF compared to urea-derived RRF. Iohexol-derived RRF did not differ from urea-derived RRF (2.48 ± 0.3 vs. 2.64 ± 0.4 ml/min, P = 0.21). The RRF contribution to the weekly dialysis prescription (Kt/V) did not differ when iohexol-derived RRF was compared to urea-derived RRF (0.94 ± 0.1 vs. 0.93 ± 0.1, P = 0.9). Additionally, the effect of iohexol on RRF was assessed in 17 HD patients. Urea-derived RRF determinations one week after iohexol exposure did not differ from those measured one week prior to iohexol exposure (3.17 ± 0.6 vs. 2.91 ± 0.5 ml/min, respectively). Thus, renal clearance of iohexol can be an accurate and safe measure of RRF in HD patients and potentially simplify delivery of the dialysis prescription.