Insulin receptor substrate-1 (IRS-1) has an important role as an early intermediary between the insulin and IGF receptors and downstream molecules that participate in insulin and IGF-I signal transduction. Here we employed an antisense oligonucleotide (IRS-1AS) to inhibit whole-body expression of IRS-1 in vivo and evaluate the consequences of short-term inhibition of IRS-1 in Wistar rats. Four days of treatment with IRS-1AS reduced the expression of IRS-1 by 80, 75, and 65% (P < 0.05) in liver, skeletal muscle, and adipose tissue, respectively. This was accompanied by a 40% (P < 0.05) reduction in the constant of glucose decay during an insulin tolerance test, a 78% (P < 0.05) reduction in glucose consumption during a hyperinsulinemic-euglycemic clamp, and a 90% (P < 0.05) increase in basal plasma insulin level. The metabolic effects produced by IRS-1AS were accompanied by a significant reduction in insulin-induced [Ser (473)] Akt phosphorylation in liver (85%, P < 0.05), skeletal muscle (40%, P < 0.05), and adipose tissue (85%, P < 0.05) and a significant reduction in insulin-induced tyrosine phosphorylation of ERK in liver (20%, P < 0.05) and skeletal muscle (30%, P < 0.05). However, insulin-induced tyrosine phosphorylation of ERK was significantly increased (60%, P < 0.05) in adipose tissue of IRS-1AS-treated rats. In rats treated with IRS-1AS for 8 d, a 100% increase (P < 0.05) in relative epididymal fat weight and a 120% (P < 0.05) increase in nuclear expression of peroxisome proliferator-activated receptor-γ were observed. Thus, acute inhibition of IRS-1 expression in rats leads to insulin resistance accompanied by activation of a growth-related pathway exclusively in white adipose tissue.