The objective of the study was to compare in vivo estimates of Cd bioavailability in two diet materials (lettuce and durum wheat grain) with bioaccessibility estimates from three in vitro methods. For both dietary materials, the Cd was either incorporated during growth or applied topically as a soluble salt just prior to experimentation. Simulated gastric/intestinal digestion using a physiologically based extraction technique (PBET) solubilized less than 56% (lettuce) or 13% (grain) of the Cd that was either incorporated into the plant tissues during growth, or added to the plant tissues before experimentation, as Cd(NO3)2 · H2O. Amended diets could not be distinguished from incorporated diets. More of the Cd solubilized from amended lettuce than from incorporated lettuce moved to the outside of MWCO 10 kD and 25 kD dialysis sacs; no difference between the amended and incorporated diets was observed for grain. The percentage of lettuce-Cd solubilized by the PBET and sorbed by Caco-2 cells was greater for incorporated than for amended lettuce; for Cd in grain, the reverse occurred. As expected, none of the in vitro estimates of bioaccessibility were the same percentage of Cd in the lettuce or grain as was measured as bioavailable in vivo. The in vitro assays all predicted that substantially less than 100% of the Cd in the foods would be bioavailable, as was identified in vivo, and simulating intestinal selectivity improved the comparison to in vivo. Some of the in vitro assays identified subtle differences between the diets (i.e., amended vs. incorporated) that were consistent with in vivo studies, and with speculated differences in Cd speciation; this suggests their potential usefulness for the study of modifiers to dietary Cd bioavailability.