The DNA-synthetic and proliferative activities of freshly isolated, nontumorigenic C3H mouse skin cells (first passage) were lowest when the extracellular free (or ionic) calcium level was reduced to between 0.05 and 0.1 mM, whereas the extracellular free calcium level in cultures of repeatedly passaged, preneoplastic C3H/10T1/2 and MCA-C3H/10T1/2 type I mouse fetal fibroblasts had to be reduced to 0.01 mM or less before the DNA-synthetic and proliferative activities were minimal. This inhibition of DNA synthesis and cell multiplication by calcium deprivation was rapidly reversed by returning the extracellular calcium level to its normal value. In contrast, the neoplastic fibrosarcoma-forming, MCA-C3H/10T1/2 type III mouse fetal fibroblasts could synthesize DNA and could multiply indefinitely even in the presence of an extremely low concentration of extracellular free calcium. Thus, the extracellular calcium requirement for DNA synthesis and proliferation appears to reflect the tumorigenic potential of the cell.