This article describes the synthesis of 2,4-di- and 2,3,4-trisubstituted benzimidazo[1,2-alpyrimidine derivatives which were tested for their cytotoxicities and their spectral characterizations by IR, H-1- and C-13-NMR, El-MS. The fused title compounds were formed by the reaction of 2-aminobenzimidazole with beta-keto esters or 1,3-alkanediones in PhMe and MeOH or in PPA, respectively, by one-pot cyclocondensation reactions. Complete structural assignments of 2,4-di- and 2,3,4-trisubstituted benzimidazo[1,2-alpyrimidine derivatives were established by IR, H-1- and 13C-NMR and EI-MS spectroscopic investigations. Single-crystal diffraction analysis was performed for 6. The cytotoxicities of the synthesized compounds on the non-cancer cells (F2408) and cancer cells (5RP7) were evaluated.