We previously reported that acetaldehyde increases the production of type I collagen in cultured rat fat-storing cells. We studied the regulation of this effect by determining the expression of procollagen type I, fibronectin and transforming growth factor-β1 messenger RNAs in passage 1 and 2 cultures of fatstoring cells exposed to acetaldehyde for up to 24 hr. By 6 hr, acetaldehyde increased the steady-state levels of α1 procollagen type I messenger RNA 3.2-fold and of fibronectin messenger RNA 2.8-fold above control values. At 24 hr, messenger RNA levels remained elevated. In contrast, transforming growth factor-β1 messenger RNA steady-state levels remained unaltered by 6 hr, but increased 1.5-fold by 24 hr. Cycloheximide (0.3 mmol/L) completely inhibited the acetaldehyde effect when added at zero time but was less effective when added at 15 min. The effect of acetaldehyde was not modified when cells were cultured in the presence of the acetaldehyde dehydrogenase inhibitor cyanamide (100 μmol/L). Fat-storing cells were also cultured in the presence of lactate (5, 15 and 25 mmol/L) for 6 hr. At none of these concentrations was any effect seen on either α1(I) procollagen or fibronectin messenger RNAs. In the presence of methylene blue, a scavenger of reducing equivalents, the effect of acetaldehyde on α1(I) procollagen and fibronectin gene expression was totally inhibited. Transcription run-on assay showed that acetaldehyde increased both procollagen type I and fibronectin transcriptional activity threefold and 2.5-fold, respectively. We conclude that acetaldehyde increases α1(I) procollagen and fibronectin gene expression through enhanced transcription by a mechanism dependent on newly synthesized proteins. Our data suggest that this effect requires no further metabolism of acetaldehyde, but is possibly caused by adduct formation. (HEPATOLOGY 1991;13:758–765.)