In 1996, the U.S. Food and Drug Administration approved olestra, a fat substitute, for use in snack foods. Previous studies had shown that olestra consumption could reduce absorption of carotenoids and fat-soluble vitamins. To determine the association between consumption of olestra-containing snack foods and serum concentrations of carotenoids and fat-soluble vitamins in a free-living population, we interviewed independent population-based cross-sectional samples of 1043 adults before olestra was available and 933 adults 9 mo after olestra snacks were introduced into the marketplace in Marion County, IN, the first major test market for olestra. A cohort composed of 403 adults from the first survey, oversampling those most frequently reporting olestra consumption during follow-up telephone interviews, completed a second survey. We assessed diet, lifestyle factors and olestra consumption, and collected blood for assays for the serum concentrations of six carotenoids, four fat-soluble vitamins and lipids. Nine months after the introduction of olestra into the marketplace, 15.5% of Marion County residents reported consuming an olestra-containing snack in the previous month, with a median frequency among consumers of 3.0 times per month. There were no significant associations or consistent trends for decreased serum carotenoids or fat-soluble vitamins associated with olestra consumption, although cohort members consuming >/=2 g/d of olestra had adjusted total serum carotenoids 15% lower compared with baseline. There were increases in serum vitamin K concentrations associated with olestra consumption (P = 0.03 in the cross section and P = 0.06 in the cohort). In summary, there was no statistically significant evidence in this free-living population of associations between olestra consumption and decreased serum concentrations of carotenoids and fat-soluble vitamins.