A 128-member specimen pooling scheme for acute HIV infection (AHI) detection was evaluated using 21 AHI specimens (range, 1,520 to 500,000 copies/ml) previously identified by RNA testing of 16-member plasma pools. HIV-1 RNA was detectable in 128-member pools for all 21 specimens; however, one pool created from a specimen with 1,827 copies/ml was nonreactive in one of three replicates.