Studies have been underway to evaluate the genetic variation in iron nutritional quality of the green leafy vegetable Amaranthus. Initial screening of 35 lines of amaranth from 12 species indicated wide variation in total iron, and small, but significant, differences in bioavailable iron, as determined by an in vitro assay. To verify if the differences in bioavailable iron detected by the in vitro assay were biologically significant, two lines of amaranth, A. tricolor Ames 5113 and A. hypochondriacus Ames 2171, were evaluated using a hemoglobin repletion assay in rats. Weanling Sprague-Dawley rats were made anemic by feeding an ironfree casein-based diet for 4 weeks. The anemic animals were fed treatment diets in which all Fe was provided by the amaranth lines. Hemoglobin levels were measured at the start and end of the treatment period to determine bioavailability. Although A. tricolor contained a higher concentration of total iron (670 ppm), the bioavailability of this iron to rats was lower than from the A. hypochondnacus line (total Fe = 210 ppm). Similar amounts of either amaranth line added to the diet produced similar changes in hemoglobin, although total iron concentrations were significantly different, confirming results observed with in vitro assays.