Abstract Purpose: To determine whether one vs two 24-hour urine collections is optimal in the metabolic evaluation of nephrolithiasis. METHODS We retrospectively reviewed all 24-hour urine collections performed at our tertiary stone clinic from July 1997 to February 2012. We identified patients with two 24-hour urine collections performed ≤10 days apart. Samples were analyzed by an outside laboratory for the standard urinary parameters. For each parameter, pairwise t tests were performed and Pearson correlation coefficients were calculated to compare samples 1 and 2. In addition, the number of cases that changed from normal to abnormal or vice versa was also evaluated for each parameter and the Kappa statistic was calculated. RESULTS A total of 813 subjects submitted two 24-hour urine collections ≤10 days apart. Mean age was 53.2 years, and mean body mass index was 28.8 kg/m(2). Based on creatinine 24/kg, subset analysis was performed for all properly collected samples (n=236). Using pairwise t test, 24-hour urine volume (P=0.0365) and phosphorus (P=0.0387) showed a statistically significant difference between samples 1 and 2. None of the other urinary parameters demonstrated a statistically significant difference when means were compared (pairwise t test, P>0.05), (range 0.061-0.9983). Pearson correlation demonstrated a high degree of correlation between two 24-hour urines for all variables (r=0.66-0.95, each P<0.0001). Depending on the urinary parameter assessed, 5.5% to 44.9% of patients changed from normalcy to abnormality, or vice versa. CONCLUSIONS A single 24-hour urine collection may have changed clinical decision making in up to 45% of patients. Therefore, we recommend two collections to optimize the diagnostic yield and appropriately target stone prevention strategies.