Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at 70°C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at 70°C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of 1,000 copies/ml at CPAL, 148 (72%) of the results varied by 0.20 log₁₀ when tested at Hershey and none varied by >0.50 log₁₀. However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log₁₀ when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log₁₀ while the maximum interrun variation was 0.52 log₁₀. High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log₁₀, while the maximum interrun variation was 0.55 log₁₀. In our patient population, a change in bDNA HIV-1 RNA results of 0.50 log₁₀ over time most likely represents normal laboratory test variation. However, a change of >0.50 log₁₀, especially if the results are >1,000 copies/ml, is likely to be significant.