Granzyme B–independent cytotoxicity is accounted for by the Fas pathway and a distinct perforin-dependent mechanism. CTLs (H-2^b anti–H-2^d) produced in day 5 MLR splenocyte cultures from wild-type (B^+/+), granzyme B^−/−, granzyme B^−/− × gld/gld, perforin (P) ^−/−, and P^−/− × gld/gld were tested against allogeneic TA3 cells at various E:T ratios after prolonged incubation (10 hours) in ⁵¹Cr release (A) and ¹²⁵IUdR release (B) assays. P^−/− × gld/gld CTLs do not exhibit any cytotoxicity even after prolonged incubation. Similar results were obtained using P815 and YAC-1 target cell lines (data not shown). Error bars show the range of values at each point. This experiment represents one of three with similar results.