Objective: Elevated urine albumin to creatinine ratio (ACR) of >30 mg/gm is a widely agreed upon indicator of pathologic albuminuria in children. However, the most reliable specimen to measure ACR in children remains undefined. We assess the range and limits of upright and supine total albumin and ACR in healthy children. Methods: Healthy children age 6 - 18 years completed 24-hour and split upright and supine urine collections. Upright, supine and 24-hour protein, albumin and creatinine were measured. Primary outcomes are range and variation in urine albumin by diurnal status, age, gender, BMI percentile and Tanner stage. Results: In healthy children, with mean age 12.9 year (sd 3.2), upright ACR was 2-fold greater than supine (13.9 vs 6.8 mg/gm, p = 0.02). The range of ACR was much greater in the upright (2 - 323 mg/gm) compared to the supine (1.7 - 76 mg/gm) samples. The average total 24-hour urine albumin was 8.4 mg (sd 9.8) and the mean ACR was 8.9 mg/gm (sd 11.7). The 24-hour albumin increased with age and Tanner stage, but this relationship was not significant after adjusting for BSA or urine creatinine. A supine or upright ACR of >30 mg/gm was found in 5.4% of each group. However, in all subjects with an elevated ACR on an individual upright or supine sample, a second 1st am ACR sample was normal. Conclusions: In healthy children there is a marked diurnal variability in ACR with a higher value from a daytime sample compared to 1st morning specimen. Screening for pathologic albuminuria should always use a first morning urine specimen.