The role of hyperinsulinemia in the pathogenesis of triglyceride (TG) and VLDL over-production in insulin-resistant states remains controversial. While studies in humans and animals have generally suggested that chronic hyperinsulinemia facilitates VLDL production, particularly in the presence of an abundant supply of substrate for VLDL synthesis, the majority of in vitro studies using cultured hepatocytes and hepatoma cell lines have demonstrated an acute inhibitory effect of insulin. Using radiolabeled VLDL tracers we have examined the acute effect of hyperinsulinemia on VLDL production in humans. We found a rapid suppression of plasma free fatty acid (FFA) levels in response to insulin and a consistent 50-60% insulin-induced suppression of both VLDL TG and VLDL apolipoprotein (apo) B in lean insulin-sensitive individuals. Elevation of plasma FFA levels by infusing heparin and Intralipid without hyperinsulinemia resulted in a marked increase in VLDL TG and VLDL apoB production. When the insulin-induced suppression of plasma FFA levels was prevented during hyperinsulinemia, VLDL TG production was still inhibited, although to a lesser extent than with insulin alone. We concluded from these findings that insulin suppresses VLDL production in insulin-sensitive humans partly by suppressing plasma FFA levels and partly by a non-FFA-mediated (perhaps direct hepatic) mechanism. In addition, we found that chronically insulin-resistant hyperinsulinemic obese individuals were resistant to this suppressive effect of insulin on VLDL apoB production, in keeping with similar findings by others performing in vitro experiments using cultured hepatocytes isolated from insulin-resistant or hyperinsulinemic rats. The relevance of these findings to the mechanism of hypertriglyceridemia associated with chronic insulin-resistant states in humans remains a matter of speculation. One hypothesis is that resistance to the normal suppressive effect of insulin, in association with other metabolic abnormalities associated with insulin resistance, may contribute to postprandial and postabsorptive hypertriglyceridemia.