Background: Programmed cell death (PCD) is characterized by a sequence of tightly regulated events that result in the activation of caspases and in internucleosomal DNA cleavage. Late apoptotic events such as DNA-strand breaks can be assayed by in situ end labeling (ISEL) and DNA measurement (sub G1) using flow cytometry. Phosphatidylserine (PS) redistribution from the inner plasma membrane leaflet to the outer leaflet, an early event in PCD, can be detected by annexin V (AxV) binding to PS. AxVfluorescein isothiocyanate (FITC) fluorescence intensity is variable and characterizes different cell populations, denoted here as AxV-negative (AxV neg ), AxV-low-positive (AxV lo ), and AxV-high-positive (AxV hi ). Methods: We investigate the correlation of three methods (ISEL, sub G1 DNA content, and AxV assay) for detecting apoptosis with focus on differences between populations with different levels of PS. We also examined the expression of PCD-regulating Bcl-2 family members in these cell populations by reverse transcription-polymerase chain reaction (RT-PCR). Chronic lymphocytic leukemia (CLL) cells exposed to fludarabine (FAMP) were used as an in vitro model. Cells with different PS/AxV levels were separated using fluorescence-activated cell sorting (FACS). Results: Only purified AxV hi cells had high positivity in the ISEL and sub G1 assays (94 Ϯ 0.6%, 88.6 Ϯ 6.6%, and 98.6 Ϯ 0.6%, respectively), indicating that late apoptotic cells are detected equally by all three methods. In the AxV lo population, ISEL was positive in 21% Ϯ 13% and DNA sub G1 in 20% Ϯ 6.6% of cells, suggesting that AxV identifies early apoptotic cells better than the other assays. Anti-apoptotic Bcl-2 and Bcl-X L were upregulated by FAMP when cells entered apoptosis (AxV lo ), as was pro-apoptotic Bcl-X S , which was undetectable in nonapoptotic AxV neg cells. Pro-apoptotic Bax was only expressed in AxV neg and AxV lo cells. Late apoptotic AxV hi cells did not express Bcl-X S or Bax. Results: (1) AxV staining is more sensitive than sub G1 or ISEL in detecting early apoptotic cells; (2) only late apoptotic cells are equally detected by all assays; (3) AxV is a valuable tool in the detection and isolation of apoptotic cells at different stages of PCD; and (4) pro-apoptotic Bcl-X S and Bax are expressed at early, not late, stages of apoptosis.