In this present study, essential oils from fruit and fruitless herba of Vitex agnus castus L. collected from Izmir, Turkey, were obtained by hydrodistillation using Clevenger-type apparatus for 3 h. The cytotoxic and apoptotic activity of each essential oil against C6, A549 and MCF 7 cancer cell lines was evaluated in vitro by four methods. The dose-dependent cytotoxic effects of the essential oils were tested on C6, A549 and MCF 7 cancer cell lines by using MU assay. According to WIT assay, the inhibitory effects of DNA synthesis, the apoptotic effect and the caspase-3 activation effects of each essential oil were determined by using different methods. The chemical composition of both the essential oils was analyzed by GC and GC/MS systems, simultaneously. Forty-one and thirty-nine volatile compounds were identified in both the fruit and fruitless herba essential oils representing 93.8% and 85.2% of the oils, respectively. Sabinene (22.7%, 16.9%), 1,8-cineole (18.2%, 22.4%) and (Z)-beta-farnesene (16.4%, 7.5%) were found to the major compounds in both the essential oils of the fruits and fruitless herba, respectively. The fruit essential oil showed higher cytotoxic effect on all tested cancer cell lines. MCF 7 cell lines were found to be the most sensitive to both the essential oils than the others. The fruit essential oil was found to be inducing apoptosis on both of A549 and MCF 7 cell lines. (C) 2014 Elsevier B.V. All rights reserved.