Anaplastic large-cell lymphoma (ALCL), as currently defined, includes a subset of tumors that have abnormalities of chromosome 2p23 (alk gene) resulting in overexpression of anaplastic lymphoma kinase (ALK). We have previously shown differences in apoptotic rate and expression of apoptosis-related proteins between ALK-positive and ALK-negative ALCL. In this study, we assessed for activated caspase-3 (aC-3), an executioner of apoptotic cell death, in ALCL cell lines and tumors. We used the Karpas 299 and SU-DHL-1 cell lines, and the caspase inhibitors Boc-D-FMK and DEVD-FMK to investigate the role of caspase-3 activation in tumor cell death after treatment with doxorubicin. Cell viability and apoptosis were assessed by trypan blue and Annexin-V methods. A caspase-3 assay was used to evaluate caspase-3 enzymatic activity. Caspase-3 activity was significantly increased in Karpas-299 and SU-DHL-1 cells treated with doxorubicin, but remained as low as control levels with addition of Boc-D-FMK or DEVD-FMK. Expression of aC-3 was also assessed immunohistochemically in 57 ALCL tumors. The mean percentage of aC-3 positive tumor cells was 3.2% in ALK-positive ALCL compared with 1.2% in ALK-negative ALCL (P=0.0003, Mann-Whitney test), and inversely correlated with BCL-2 expression (P=0.01, Mann-Whitney test). aC-3 expression did not correlate with patient outcome in either the ALK-positive or ALK-negative ALCL groups. In conclusion, doxorubicin-induced cell death of ALK-positive ALCL cells involves caspase-3 activation in vitro. aC-3 levels correlate with ALK expression in ALCL tumors.