Population-wide variation in genomic RNA of human immunodeficiency virus type 1 (HIV-1) encompassing the V3 loop of the envelope protein was studied in serum samples of 74 newly infected individuals from three Dutch cohorts: 30 homosexual men, 32 drug users, and 12 hemophiliacs. During acute infection, HIV-1 RNA sequences present in serum are relatively homogeneous, which makes direct sequencing feasible. This offered an opportunity to study the infecting virus variants before mutations had accumulated in the new host. The sampling dates ranged from 1980 to 1991, thus spanning the entire AIDS epidemic in The Netherlands. The diversity in the sequenced region increased over time in both the homosexual and the drug-user risk groups. Furthermore, this increase was associated with an increase in antigenic variation, as witnessed by serum reactivity to a V3 peptide panel. Despite this diversification, some 1990 sequences still closely resembled the earliest 1980 sequence, making ancestral inferences problematic. No evidence was found of a change in the master sequence of the virus quasi-species over time. At the amino acid level, no risk-group-associated variation was found, but at the nucleotide level, the drug-user and homosexual/hemophiliac sequences could be distinguished on the basis of a single silent nucleotide change in the sequence encoding the tip of the V3 loop. Hemophiliac sequences could not be distinguished from those of homosexuals. In spite of the large and increasing genetic variability, all sequences were more similar to the European/American HIV consensus sequence than to that of non-Western strains.