The two major human gammadelta T cell subsets, Vdelta1 and Vdelta2, display differences in tissue tropism and agonist responses, but we have little insight into global differences that may exist at the gene expression level. This is due to the small numbers of these cells that can be obtained from healthy donors, which limit comprehensive, comparative gene expression analyses. We established a culture method that expands Vdelta1 and Vdelta2 cells from the same PBL preparation to levels sufficient for sorting and microarray analysis. Although the subsets were expanded identically (anti-TCR mAb, plus IL-15), 392 and 614 genes were identified, which were differentially expressed in the two subsets, from two donors, respectively. Approximately 4500 genes changed in both subsets following PMA/ionomycin treatment; about 50% of these genes were subset-specific. Both subsets responded to a crude LPS preparation, but only 6% of the responsive genes were the same. The differentially expressed genes were consistent with Vdelta2 cells being more inflammatory and Vdelta1 cells having more of a regulatory phenotype. Both subsets expressed transcripts encoding an array of innate and NK cell receptors, supporting the relationship of gammadeltaT cells to the innate immune system. Our results indicate that circulating Vdelta1 and Vdelta2 subsets in humans have considerable inherent differences in gene expression following treatment with the same agonist. The patterns of differentially expressed genes are consistent with unique functional roles for these cells in vivo.