A limited number of MHC class I-b molecules have been shown to present pcptidcs to T cells. Among these molecules is the T23 encoded protein, Qa-l>, which is recognized by αβ and γδ T cells. Qa-1b is widely expressed in mouse tissues and its predicted peptide binding groove is similar to that of other class I molecules. However, only two such peptides have been described, i.e., a co-polymer Gluso-Tyr50 that is presented to a γδ T cell hybridoma and a nonamer derived from the leader peptide of several class 1 molecules (Qdm) that is recognized by Qa-1b-specific CTL. To lest the hypothesis that Qa-1b molecules are involved in presentation of peptides derived from intraccllular bacteria. We incubated mycobacterial sonicate (MS) with WEHI 164 cells, we found that MS substantially enhanced lysis of these cells by Qa-1-specific CTL. This enhancement was Qa-1-specific since neither Kb-rcstricled nor MS-resiricied lysis was affected by the same treatment. Extraction of MS with chloroform/methanol, a known Hpid extradant, revealed that most of the enhancing activity is in the organic phase. In addition, treatment of MS or the organic phase extract with proteinases such as trypsin, proteinase K or pronase did not decrease this enhancing activity. However, incubation of any of these proteinases with ND1 (M3-binding peptide) or OVA peptides abolished their sensitization activity. Furthermore, MS or the organic phase extract sensitized RMA-S cells for killing by Qa-1b-specific CTLs. Taken together, these findings indicate that Qa-1b molecules present a proteinase-resisiant hydrophobic mycobacterial antigen to Qa-1-specific CTL. The biochemical nature of this antigen is under investigation. Supported in part by NIH grant AII8882.