An experiment was conducted to evaluate the effect of type of corn endosperm on nutrient digestibility in lactating dairy cows. Near-isogenic variants of an Oh43 x W64A normal dent endosperm hybrid carrying floury-2 or opaque-2 alleles were grown in spatial isolation in field plots and harvested as dry shelled corn. Six ruminally cannulated, multiparous Holstein cows (67 +/- 9 d in milk at trial initiation) were randomly assigned to a replicated 3 x 3 Latin square design with 14-d periods; the first 11 d of each period were for diet adaptation followed by 3 d of sampling and data collection. Treatment diets that contained dry rolled vitreous-, floury-, or opaque-endosperm corn [33% of dry matter (DM)], alfalfa silage (55% of DM) and protein-mineral-vitamin supplement (12% of DM) were fed as a total mixed ration. The percentage vitreous endosperm was zero for floury and opaque endosperm corns and 64 +/- 7% for the vitreous corn. Prolamin protein content of floury and opaque endosperm corns was 30% of the content found in vitreous corn. Degree of starch access and in vitro ruminal starch digestibility measurements were 32 and 42% greater on average, respectively, for floury and opaque endosperm corns than for vitreous corn. Dry matter and starch disappearances after 8-h ruminal in situ incubations were, on average, 24 and 32 percentage units greater, respectively, for floury and opaque endosperm corns than for vitreous corn. Ruminal pH and acetate molar percentage were lower, propionate molar percentage was greater, and acetate: propionate ratio was lower for cows fed diets containing floury and opaque endosperm corns than for cows fed vitreous corn. In agreement with laboratory and in situ measurements, total-tract starch digestibility was 6.3 percentage units greater, on average, for cows fed diets containing floury and opaque endosperm corns than vitreous corn. Conversely, apparent total-tract neutral detergent fiber (NDF) digestibility was lower for cows fed diets containing floury and opaque endosperm corns compared with vitreous corn. The type of endosperm in corn fed to dairy cows can have a marked effect on digestion of starch and NDF. Feeding less vitreous corn increased starch digestion but decreased NDF digestion.