Background: Glomerular filtration rate (GFR) estimation equations have never been validated in the HIV population. This pilot study aimed to compare all currently available methods of kidney function assessment with nuclear GFR in HIV-infected adults. Methods: Patients underwent GFR measurement with 99mTc-diethylenetriaminepentaacetic acid (Tc-99m Pentetate), and measured values were compared with results of creatinine-based estimation equations [abbreviated 4-variable Modification of Diet in Renal Disease (MDRD) formula and Cockcroft-Gault (CG) formulae], 24-hour urine creatinine clearance and estimated cystatin C GFR. Results: Twenty-seven HIV-infected adults were studied. Most were male and Caucasian, with a mean age of 52 years. Median CD4 was 290 cells/mm3, 70% of patients had HIV RNA <50 copies/ml and all were receiving highly active antiretroviral therapy (median 5 drugs). Median Tc-99m Pentetate-GFR was 91 ml/min/1.73 m2. Despite greater bias and similar accuracy, the MDRD formula was more precise than the CG formula, regardless of whether CG estimations were corrected for ideal body weight or body surface area. Relative accuracy within 30% of nuclear GFR was greater for the MDRD formula than for all other methods. The performance of 24-hour urine creatinine clearance was similar to that of the MDRD formula for patients with GFR <90 ml/min/1.73 m2, although it performed less well at higher GFR. The performance of cystatin C GFR was inferior to that of all the creatinine-based methods. Conclusions: While no method of kidney function estimation performed highly, both 24-hour urine creatinine clearance and the MDRD formula performed with a level of precision and accuracy sufficient for clinical decision making. Our findings support the preferential use of the MDRD formula in the treated HIV population and suggest that there are no HIV-specific factors that limit equation applicability. Larger validation studies are needed to confirm our findings and allow generalization to the HIV population at large.