We have compared the cytolytic activities and the cellular compositions of the intestinal intraepithelial lymphocyte (i-IEL) populations in three different combinations of conventional (CV) and germ-free (GF) mice. Cytolytic activity of i-IELs expressing gamma delta T-cell antigen receptors (TCRs) is strain dependent in CV mice (high vs. low), and this strain-dependent variability is unaltered in the GF condition. Although absolute numbers of gamma delta i-IELs are slightly decreased, the composition of CD8 alpha alpha+ and CD4-CD8- subsets and the usage of TCR gamma- and delta-chain variable gene segments by gamma delta i-IELs remain the same in GF mice. By contrast, cytolytic activity of alpha beta TCR-expressing i-IELs is uniformly high in CV mice but attenuated sharply in the GF condition. A conspicuous decrease in the total numbers of alpha beta i-IELs is also noted, and CD8 alpha beta+ and CD4+CD8+ subsets are reduced, whereas the CD8 alpha alpha+ subset is expanded in GF mice. These results indicate that microbial deprivation preferentially influences the alpha beta i-IEL population to decrease and become noncytolytic but has little effect on the pool size or characteristics of gamma delta i-IELs. Consequently, cytolytic activity of freshly isolated i-IELs from GF mice is determined by T cells expressing gamma delta TCRs and is found to be strain dependent.