Phase-variable lipoproteins are commonly found in Mycoplasma species. Mycoplasma pulmonis contains a family of extensively studied phase- and size-variable lipoproteins encoded by the vsa locus. The Vsa surface proteins vary at a high frequency, the in vivo significance of which has yet to be determined. We investigated the role of Vsa phase variation in respect to tissue tropism and avoidance of the immune system in the mouse host. Mycoplasmas were cultured 3, 14, and 21 days postinoculation from the nose, lung, trachea, liver, and spleen of experimentally infected C57BL/6 (wild-type), C5TBL/6-RAG-1 -/- (RAG-/-), and C57BL/6-inducible nitric oxide synthase (iNOS)-/- (iNOS-/-) mice. In wild-type and iNOS -/- mice, a large number of Vsa variants were seen by 21 days postinoculation. In contrast, little Vsa variation occurred in all tissues of RAG-/- mice. Analysis of isolates from 14 days postinoculation revealed less variation of the Vsa proteins in iNOS-/- mice than in the wild type. Western blot analysis of isolates from each strain of mouse demonstrated that Vsa phase variation occurred independently of size variation, indicating no obvious selection pressure for size variants. Additionally, these experiments provided no evidence that mycoplasmas producing particular Vsa proteins adhered only to specific tissues. The data strongly indicate that Vsa phase variation is a mechanism for avoidance of the immune system with no obvious contribution to tissue tropism. Copyright © 2005, American Society for Microbiology. All Rights Reserved.