The extractability and distribution of manganese (Mn) and zinc (Zn) were evaluated in acidic agricultural soils from Central Spain. Both single (0.1 M hydrochloride [HCl] and 0.05 M ethylenediaminetetraacetate [EDTA]) and sequential extraction procedures (SEP) (modified Tessier procedure and Community Bureau of Reference [BCR] protocol) were applied to 29 representative soils that belong to the Alfisol, Inceptisol, and Entisol orders. Average relative Mn extractabilities with respect to the total content (16.6% for HCl and 31.9% for EDTA) were higher than those of Zn (7.7% for HCl and 6.5% for EDTA). Manganese was mainly released in the oxide-bound phase of both SEP (33.1% for modified Tessier and 48.9% for BCR), whereas Zn was predominantly found in the residual fraction (49.1% for modified Tessier and 31.4% for BCR). Significant correlations were only found between the amounts of extractable Zn and the oxide-bound fraction in both SEP. Few relationships were established between Zn fractions extracted by the BCR procedure and those obtained with the Tessier method. Both metal concentrations in spring barley (Hordeum vulgare L., cv Beka) grown in 11 selected soils and the calculated soil/plant transfer coefficients (soil/plant concentration factor (CF), mean values of 31.2 for Mn and 196 for Zn) were poorly (or not at all) correlated with the different extracted soil fractions (single and sequential). A number of empirical equations have been obtained by regression analyses to predict the Mn and Zn uptake by barley, with soil metal forms and some soil characteristics as components (pH for Mn and organic matter for Zn). Values of R2 in the equations were relatively low (<68%). Single-extraction techniques produced worse results than SEP for the evaluation of Mn phytoavailability. The modified Tessier procedure provided better predictions of Zn uptake by plants than BCR, but not better than those obtained with the HCl extraction method.