Previously, CD8⁺ T cells were found to be a sensitive target for suppression by Δ⁹-tetrahydrocannabinol (Δ⁹-THC) in a murine model of influenza infection. To study the effect of Δ⁹-THC on CD8⁺ cytotoxic T lymphocytes (CTL), an allogeneic model of MHC I mismatch was used to elicit CTL. In addition, to determine the requirement for the cannabinoid receptors 1 (CB₁) and 2 (CB₂) in Δ⁹-THC-mediated CTL response modulation, mice null for both receptors were used (CB₁ ^−/−CB₂ ^−/−). Δ⁹-THC suppressed CTL function independent of CB₁ and CB₂ as evidenced by reduction of ⁵¹Cr release by CTL generated from CB₁ ^−/−CB₂ ^−/− mice. Furthermore, viability in CD4⁺ and CD8⁺ cells was reduced in a concentration-dependent manner with Δ⁹-THC, independent of CB₁ and CB₂, but no effect of Δ⁹-THC on proliferation was observed, suggesting that Δ⁹-THC decreases the number of T cells initially activated. Δ⁹-THC increased expression of the activation markers, CD69 in CD8⁺ cells and CD25 in CD4⁺ cells in a concentration-dependent manner in cells derived from WT and CB₁ ^−/−CB₂ ^−/− mice. Furthermore, Δ⁹-THC synergized with the calcium ionophore, ionomycin, to increase CD69 expression on both CD4⁺ and CD8⁺ cells. In addition, without stimulation, Δ⁹-THC increased CD69 expression in CD8⁺ cells from CB₁ ^−/−CB₂ ^−/− and WT mice. Overall, these results suggest that CB₁ and CB₂ are dispensable for Δ⁹-THC-mediated suppression and that perturbation of Ca²⁺ signals during T cell activation plays an important role in the mechanism by which Δ⁹-THC suppresses CTL function.