Con A splenic lymphoblasts were incubated with phosphatidyl-inositol specific phospholipase C (PIPLC) derived from Bacillus thuringiensis and subsequently analyzed for Qa-2 Ag with the Qa-2 reactive mAb Qa-m2. This treatment completely removed Qa-2 detectable Ag on lymphoblasts from H-2d animals, indicating that these molecules are likely anchored to the cell membrane through phosphatidyl inositol (PI). Although exposure of lymphoblasts from H-2b mice to PIPLC greatly reduced Qa-2 expression, a subpopulation of cells retained a limited quantity of the Ag. Bulk cultured anti-Qa-2 CTL generated against the Qa-2 region from H-2b haplotype mice lysed Qa-2+ targets from B6.K2 (H-2b) and BALB/cJ (H-2d) animals. Pretreatment of these lymphoblast targets with PIPLC completely abolished lysis of the BALB/cJ target cells, whereas lysis of B6 targets was reduced only slightly. Anti-Qa-2 CTL clones tested against PIPLC-treated B6 target cells revealed two patterns of reactivity. One group of clones was unaffected in its ability to lyse PIPLC-pretreated targets and cross-reacted on Q6d/Ld molecules expressed on transfected L cells. A second group was unable to lyse PIPLC-pretreated lymphoblasts and cross-reacted on Q7d/Ld targets. These data suggest that H-2b-derived lymphoblasts express two different types of Qa-2 molecules with respect to PIPLC sensitivity; one type is sensitive to PIPLC and cross-reactive with Q7d, the other type is resistant to PIPLC and cross-reactive with Q6d. In contrast, H-2d lymphoblasts express only the PIPLC-sensitive type of molecules. It was also noted that bulk cultured anti-Qa-2 CTL more readily lysed H-2b target cells expressing a smaller quantity of PIPLC-resistant Ag than H-2d targets expressing a larger amount of PIPLC-sensitive Ag. Further, anti-Qa-2 CTL clones readily lysed PIPLC-treated target cells expressing very low levels of serologically detectable Qa-2. This suggests that recognition of class I molecules anchored to the membrane via a PIPLC-resistant linkage may more readily activate CTL for expression of lytic activity than molecules anchored through PI.