Ether phospholipids are analogs of the naturally occurring 2-lysophosphatidylcholine that have been reported to have selective in vitro/in vivo antitumor activity. Their antiproliferative effect has been found against a variety of animal and human tumor cell lines. We have characterized the cytostatic activity of four ether phospholipids, the methoxy-substituted edelfosine (ET-18-OCH 3), the thio-derivative ilmofosine (BM 41.440), and two new aza-alkylphospholipids, BN 52205 and BN 52211, on a human tumor cell line derived from a colon adenocarcinoma, the HT29. A flow cytometric approach has been used and, contrary to previous studies, longer treatment times have been performed to allow multiple cell population doublings. The results confirm that the cytostatic activity of the four ether phospholipids is characterized by multiple "terminal points", as the drugs' action results in a G1 block, a slowdown of the transition from late-S to G2, followed by an accumulation of HT29 cells in the G2 phase of the cell cycle. Tumor cells in late G1 at the time of treatment progressed through S before being blocked in G2. In a similar fashion, tumor cells in late G2 at the time of treatment went through M but were then halted in G1. The long-term treatment studies indicate that the ether phospholipid cytostatic activity is partially reversible, depending on the drug concentration and the duration of the treatment.