Human diploid fibroblasts, arrested following serum or mevalonate depletion, were restimulated to a maximal rate of DNA synthesis within 24 h after the addition of serum or mevalonate, respectively. In both cases the initiation of DNA synthesis was preceded by a 12-h prereplicative phase. Upon the stimulation with serum there was a rapid increase in HMG-CoA reductase activity, reflecting an elevated formation of mevalonate, which reached its maximal value 4 h after serum replenishment. If this serum-induced increase in mevalonate synthesis was inhibited, the subsequent initiation of DNA synthesis was prevented. Serum stimulation also increased the level of N-linked glycosylation, an event that was dependent on the increase in HMG-CoA reductase activity. After treatment of the cells with tunicamycin, an inhibitor of N-linked glycosylation, they failed to enter the S-phase. However, an increased level of N-linked glycosylation was not required during the whole of the period after serum stimulation. Instead, it seemed to be of critical importance only during the mid stage of the prereplicative phase (i.e., 4-8 h after stimulation). Our data suggest that the N-linked glycosylation required for initiation of DNA synthesis is of high-molecular-weight (90-240 kDa) proteins. These high-molecular-weight glycoproteins may include growth factor receptors. Indirect evidence raises the possibility that the expression of growth factor receptors may play a regulatory role in the mevalonate-dependent growth activation of human fibroblasts.