Cytotoxic T lymphocytes (CTLs) lyse target cells by secreting the contents of granules onto target cell membranes. These granules contain perforin, granzymes and various lysosomal enzymes. CTLs themselves are highly resistant to perforin-mediated lysis. Other target cells manifest differing degrees of susceptibility to perforin. We have examined the influence of surface membrane proteins on the interaction of perforin with target membranes. Surface proteins on A20 and YAC murine cell lines were digested with various proteases. Treatment with proteases causes these tumor cells to become much more sensitive to perforin. A mixture of granzymes derived from CTL granules did not enhance the sensitivity of target cells to perforin, although comparable trypsin activity markedly affected sensitivity to perforin. We also investigated whether surface proteolysis mitigated resistance of CTLs to perforin attack. CTLL-2 remained resistant to granule-mediated lysis despite pre-incubation with pronase. These experiments demonstrate that surface proteins interfere with the interaction of perforin with membrane bilayer, possibly due to steric hindrances. They also show that the factor(s) protecting CTLs from perforin are inaccessible to proteases outside the cell and may not be exposed on the surface Infection with the human immunodeficiency virus (HIV) results in profound defects in the immune system, most prominently loss of CD4+ T-lymphocytes. The goal was to determine whether the activity of granzyme A and B have been altered in HIV-infected hemophiliacs. A colorimetric assay that measures cleavage of a synthetic substrate, N$\alpha$-benzyloxycarbonyl-L-lysine thiobenzyl ester, was used to quantitate granzyme A activity. Granzyme A activity in individual CD8+ T cells from HIV seropositive hemophiliacs was significantly lower than the activity in cells from HIV seronegative hemophiliacs These results indicate that individual cytotoxic cells in HIV-infected hemophiliacs have reduced activity of granzyme A, which may result in a defect of CTL-mediated cell killing in these patients. However, using QC-RT-PCR to quantify the mRNA levels of granzyme B from healthy donors and hemophiliacs, we conclude they all have similar levels of granzyme B mRNA expression. Granzyme A and B may be differentially regulated at transcription level