Polymethylene-bis(1-nitrosourea), polymethylene-bis(1-nitroso-3-nitroguanidine), and polymethylene-bis(1-nitroso-p-toluenesulfonamide) derivatives were tested for antitumor effect against rat ascites hepatoma AH-13 and mouse leukemia L-1210. Bisnitrosoureas were effective against AH-13 and L-1210, bisnitrosoguanidines were effective against AH-13 alone, and bisnitrosotoluene-sulfonamides were ineffective against both tumor lines. Of all these compounds, 1,1'-ethylene-bis(1-nitrosourea) (EBNU) was the most effective. The antitumor effect of EBNU was compared with that of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Intraperitoneal administration of EBNU according to the schedule, day 1, days 1 and 5, and days 1, 5, and 9 after intraperitoneal inoculation of L-1210 showed marked prolongation of host survival, although the effective doses used were a few times higher than those used in BCNU to obtain a similar effect. The minimum effective dose (MED) of EBNU on AH-13 cells was estimated as 1 mg/kg, which was 10 times less than that of BCNU, suggesting that EBNU was more effective than BCNU against AH-13.