Betalactam antibiotics (BLA) are the most widely used antibacterial drugs in practical medicine. Recent experiments suggested that BLA, especially after "aging" in aqueous solutions, have an inhibitory effect on the growth of a variety of cultured human cells by interfering with DNA synthesis (Neftel et al. Cell Biol. Toxicol. 2, 513-521, 1986). Our initial observation that the replicative DNA polymerase alpha might be the target of the action of betalactam compounds (Hübscher et al. Cell Biol Toxicol. 2, 541-548, 1986) is now substantiated due to the following experimental data: (i) extractable DNA polymerase alpha is greatly reduced in cells that had been treated with BLA; (ii) the relative cellular distribution of thymidine and of its phosphorylated derivatives is not affected by BLA; (iii) BLA inhibit crude and highly purified mammalian DNA polymerase alpha; (iv) the inhibitory effect appears to be of the mixed type with a slight deviation from purely non-competitive behaviour towards the four deoxyribonucleoside triphosphates and; (v) the inhibition is evident in aphidicolin sensitive DNA polymerases from mammalian tissues and in DNA polymerases from DNA viruses such as Herpes simplex and Vaccinia. In sum, the results suggest that one of the most commonly used class of drugs has a target within eukaryotic cells being most likely the replicative DNA polymerase alpha.