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Due to the extraordinary degree of genetic diversity of HIV-1 and the structural complexity of its envelope glycoproteins, designing an effective vaccine is difficult, requiring the development of viral reagents to assess vaccine-elicited neutralizing antibodies. The aim of this study was to improve on our previously developed panel of HIV-1 strains of different genetic forms, focusing on strains from acute and recently acquired infections as the most representative of the transmitted viruses. HIV-1 primary isolates were expanded in peripheral blood mononuclear cells. Viral stocks of 40 ml each were produced. Syncytium-inducing (SI) phenotype, coreceptor use, and TCID(50)/ml were determined. Near full-length HIV-1 genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Forty-four HIV-1 strains were included in the panel. Twenty-four (54.1%) strains were from early infections (16 acute and 8 recent); of them, 21 (87%) were sexually transmitted. NSI/R5 phenotype was detected in 37 (84.1%) viruses and SI/R5,X4 in another 7 (15.9%). TCID(50)/ml ranged between 10(4) and 10(6.6). Twelve different genetic forms constituted this panel: subtypes A1, B, C, F1, and G; circulating recombinant forms CRF02_AG, CRF14_BG, and CRF24_BG; and unique recombinant forms CRF02_AG/A3, BF1, CRF12_BF/B, and DF1G. In conclusion, in this study, we report the development of a comprehensive and well-characterized panel of HIV-1 isolates for assessing neutralization in HIV vaccine research. This panel is available for distribution through the Programme EVA Centre for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC).