Ascorbic acid positively affects the synthesis of collagen, the most abundant extracellular protein. The mechanism by which ascorbate mediates the increased synthesis is debated. One recent hypothesis suggests that ascorbic acid induces an increase in lipid peroxidation and that this increase, in some manner, up-regulates collagen gene expression. Evidence is presented that indicate increases in lipid peroxidation [thiobarbituric acid (TBA)-reactive material] is coincidental to collagen increases in ascorbate-treated cells, not a causal factor. Thus, cell impermeable iron chelators totally abolish ascorbate-mediated lipid peroxidation but do not affect collagen synthesis in the least. Decreases in TBA-reactive products seen at higher ascorbate levels (indicative of the well known pro- to antioxidant conversion of ascorbate in vitro) are paralleled by decreases in collagen synthesis. The decrease seen in collagen is completely reversed by treatment of the cell cultures with superoxide dismutase and catalase while the measure of lipid peroxidation is unaffected by coincubation with these antioxidant enzymes. Additionally, incubation conditions used to measure ascorbate induction of TBA-reactive material (buffers vs media, adherent vs detached cells) were found to be very important and results support the thesis that lipid peroxidation and collagen synthesis can be dissociated. While these results do not rule out a role for lipid mediators in regulating collagen synthesis at some level, they suggest this mechanism need not be involved for collagen increases seen in ascorbic acid-treated cells.