The main objective of this study was to develop an optimal methotrexate (MTX) microemulsion (M-MTX) and to evaluate the suppressive effect of MTX-loaded microemulsion on human breast, ovarian, and prostate carcinoma cell lines. The microemulsion was made up of corn-oil as the oil phase, a mixture of Cremophore EL and Span 80 as surfactants, and isopropyl alcohol as co-surfactant and 0.1 N NaOH as the aqueous phase. MTX (0.1 mg/mL MTX) was added into the microemulsion at the last stage. The pH of M-MTX was adjusted to pH 8 +/- 0.1 and the physicochem ical stability of the formulation was observed. The particle size distribution was measured by Zetasizer 3000 HSA. The mean droplet diameters of M-MTX were determined as 13.3 +/- 0.1 nm (poly dispersity index = 530 +/- 0.01). The antitumor effects of M-MTX were examined on human breast (MCF-7), ovarian (OVCAR), and prostate (DU 145) carcinoma cell lines. It was clearly demonstrated that M-MTX had a significant cytotoxic effect on different carcinoma cell lines and the cytotoxic effect of M-MTX was significantly more than that of commercial preparation (C-MTX) (P<0.05). According to the in vitro cytotoxicity studies, it can be concluded that when MTX was incorporated into the microemulsion (M-MTX), which is a new drug carrier system, it suppresses tumour cell growth on multiple tumor lines. These results indicate that M-MTX may be effective as an antitumor agent that induces apoptosis. Drug Dev Res 70:49-56, 2009. (C) 2009 Wiley-Liss, Inc.