The in vivo effect of insulin upon the incorporation of 32Pi into nuclear proteins was investigated in quiescent baby hamster kidney fibroblasts (BHK-21). Insulin has previously been shown to be mitogenic in these cells (Richman, R. A., Johnson, R. A., and Friedman, D. L. (1981) Proc. Soc. Exp. Biol. Med. 168, 196-203). Incorporation into two proteins (Mr = 62,000 and 72,000) in the 0.4 M salt-resistant nuclear fraction was enhanced 2-6-fold by insulin. The effect of insulin (20 nM) was observable within 5 min of treatment, reached a maximum at 15 min, and continued for at least 90 min. The half-maximal effect of insulin was obtained at a concentration of approximately 1 nM. Analysis of nuclear matrix preparations indicated that the two insulin-sensitive proteins were prominent nuclear matrix proteins and suggested that they were lamins A and C. This was confirmed by immunostaining with lamin antibodies and by two-dimensional electrophoresis. These studies indicate that insulin rapidly stimulates the incorporation of phosphate into nuclear lamins A and C in quiescent BHK-21 fibroblasts.