We previously developed a new formulation of methotrexate (MTX) that is adsorbed onto a suspension of activated carbon particles (MTX-CH) and reported the usefulness of local administration in murine tumors. The present study examines the effects of human colon carcinoma (LoVo) xenografts and the acute toxicity of MTX-CH compared with MTX aqueous solution (MTX-AQ) in mice. In therapeutic experiments, LoVo cells were implanted into the backs of BALB/c nude mice. When the cells had developed into tumors, we performed an intratumoral administration of a weekly dose of 30 mg/kg. The MTX concentration in the tumor was compared between the MTX-CH group and MTX-AQ group. In experiments on acute toxicity, MTX-CH and MTX-AQ were injected subcutaneously in BDF1 mice, and intoxication symptoms, changes in body weight, and date of death were recorded. In the therapeutic experiments, intratumoral administration of MTX-CH was much more effective in suppressing the tumor growth compared with MTX-AQ. In experiments of acute toxicity, the death time of the MTX-CH group was delayed to a greater extent, and the 50% lethal dose (LD 50 ) values of MTX-CH were lower than those of MTX-AQ. The LD 50 values of MTX-CH are 75 times higher than the efficacious dose of 30 mg/kg. The present results suggest that intratumoral administration of MTX-CH is useful for local therapy and the therapeutic dose of MTX-CH can be safely injected subcutaneously.Intratumoral administration therapy is useful for the patients with digestive tract cancer, who cannot receive surgical treatment due to a locally advanced unresectable tumor or with surgical risks such as cardiac diseases, aging, and so on (Kuwayama et al., 1984;Cascinu et al., 1998). However, locally injected drugs in aqueous solution form are rapidly absorbed through blood capillaries into the circulatory blood; therefore, they are not effective at the injected sites (Ballard, 1968). To retain the drug in the tumor for a long period, we previously developed a new formulation of methotrexate (MTX) that is adsorbed onto a suspension of activated carbon particles (MTX-CH) (Hagiwara et al., 1992(Hagiwara et al., , 1994. Activated carbon adsorbs a large amount of MTX onto the surface of particles and releases most of the adsorbed MTX into the tissues. The adsorption of the isotherm of MTX into activated carbon shows that the adsorbed MTX remains in dynamic equilibrium with the concentration of free MTX around the activated carbon particles. When the free MTX is consumed by washout or binding to tissue and the concentration of MTX in a free state decreases around the carbon particles, the carbon releases the adsorbed MTX, thus replacing the decreased concentration (Hagiwara et al., 1994). We previously reported on the effectiveness of local administration of MTX-CH in the early stages of esophageal cancers or gastric cancers. In the case of most of these patients, the primary lesion disappeared completely without any side effects (Hagiwara et al., 1996(Hagiwara et al., , 1997(Hagiwara et al.,...