ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (< 60 min) with insulin or A-II increased phosphorylation of ERK1/2 at 15 min and ERK5 at 5 min. Chronic treatment (≤ 8 h) with insulin increased ERK1/2 phosphorylation by 4 h and ERK5 by 8 h. A-II stimulated phosphorylation of ERK1/2 by 8 h and ERK5 by 4 h. The EC50 for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 nM and 0.1 nM, whereas the EC50 for A-II was 2 nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.