Pharmacological approaches employing agents that bind to the Bcl-2 surface pocket have been used successfully to neutralize the activities of anti-apoptotic Bcl-2 family members, and induce apoptosis. Several reports suggest that Bcl-2 expression/function is cell cycle-dependent. Hence, is killing by Bcl-2 surface pocket binding agents also cell cycle-dependent? In the current study, centrifugal elutriation was used to generate cell cycle phase-enriched preparations of the human myelomonocytic leukemia cell line U937. Elutriated fractions were treated with sub-optimal cytotoxic concentrations of the pro-apoptotic, non-peptidic Bcl-2 pocket-binding agent HA14-1. A concentration of HA14-1 sufficient to kill approximately 30-35% of asynchronous cultures minimally affected the progressions of elutriated post-checkpoint G1, S, G2/M phase cells, but completely suppressed the progression of pre-G1 checkpoint G1 cells. Analyses of trypan blue exclusion, morphology, nuclear condensation, mitochondrial membrane potential, sub-diploid DNA contents, and caspase-3 indicated preferential killing and induction of apoptosis in pre-G1 checkpoint G1 and G2/M phase cells.