Giardia lamblia infections are associated with antigenic variation of the parasite, which is generated by a continuous change of the variant-specific surface proteins (VSPs). Many investigations on the process of antigenic variation were based on the use of G. lamblia clone GS/M-83-H7, which expresses VSP H7 as its major surface antigen. In the present study, mice were infected with the aforementioned clonal line to investigate vsp gene expression during the complex process of antigenic variation of the parasite. Trophozoites collected from the intestines of individual animals at different time points postinfection (p.i.) were analyzed directly for their vsp gene expression patterns, i.e., without cultivating the recovered parasites in vitro. Because few trophozoites were recovered at late time points p.i., a combined 5' rapid amplification of cDNA ends-reverse transcription-PCR approach was utilized. This allowed detection and subsequent sequence analysis of vsp gene transcripts upon generation of amplified cDNA analogues. The same PCR approach was applied for analysis of vsp gene expression in variants obtained after negative selection of axenic GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an overall view of the entire panel of in vivo- and in vitro-derived parasite populations, expression of 29 different vsp gene sequences could be demonstrated. In vivo antigenic variation of G. lamblia clone GS/M-83-H7 was shown to be a continuous process involving the consecutive appearance of relatively distinct sets of vsp transcripts. During the 42-day infection period investigated, this process activated at least 22 different vsp genes. Comparative molecular analyses of the amino acid level demonstrated that all cDNA segments identified encode structural elements typical of the terminal segment of Giardia VSP. The similarity of most of the GS/M-83-H7 VSP sequences identified in the present study supports previous suggestions that vsp gene diversification in G. lamblia is the result of ancestral gene duplication, mutation, and/or recombination events.