The antigenically distinct Shiga-like toxins (SLTs) SLT-1 and SLT-II are cytotoxic for both Vero and HeLa cells and use Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb3) molecules as functional receptors. SLT-II-related variants SLT-IIvp and SLT-IIvh, produced by a porcine isolate and a human isolate, respectively, are cytotoxic for Vero but not HeLa cells. To investigate the basis for these differences in cytotoxic specificity among SLTs, the nature of the receptor for the SLT-II variants was examined. First, the patterns of binding of SLT-II and the SLT-II variants to Gb3 receptor analogs Gal alpha 1-4Gal-bovine serum albumin and Gal alpha 1-4Gal beta 1-4Glc-bovine serum albumin were compared. SLT-IIvp bound the trisaccharide neoglycoprotein preferentially, while SLT-IIvh bound both analogs equally but with less affinity than did SLT-II. Next, the glycolipids to which the SLT-II variants bound in Vero and HeLa cells were identified by thin-layer chromatography. SLT-IIvp bound to Gb3, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb4), and Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb5) in Vero cells but only Gb3 in HeLa cells. However, SLT-IIvh bound to Gal alpha 1-4Gal beta 1-1Cer (Gb2) and Gb3 in HeLa cells but only Gb3 in Vero cells. In addition, hybrid toxins (SLT-IIvp subunit A with SLT-II subunit B or SLT-II subunit A with SLT-IIvp subunit B) were used to show that the receptor specificities of the SLTs was B subunit specific. These differences in receptor specificities are important in vivo, as evidenced by a 400-fold difference in the 50% lethal doses of purified SLT-IIvp and SLT-II (200 versus 0.5 ng, respectively) for mice. These data indicate that SLT-II-cytotoxic variants can occur as a consequence of differences in receptor specificity and affinity.