The binding kinetics of native IL-3 and a set of truncated IL-3 variants to the alpha subunit of the IL-3 receptor (IL-3R alpha) were studied using surface plasmon resonance. These variants, with amino acid substitutions at residues, 22, 42, 43, 45, 46, 113, or 116, have previously been identified to have altered capacity to stimulate cell proliferation compared to native IL-3(1-133). In this study, variants E43N and F113Y exhibited > 100-fold slower association rates than IL-3(15-125) consistent with residues 43 and 113 being essential for the binding of IL-3 to the IL-3R alpha. Variants G42A, G42D, Q45V, D46S, K116V, and K116W exhibited increased association rates (up to 15-fold relative to IL-3(15-125)) and decreased dissociation rates (up to 7-fold). The results demonstrate that both the association and dissociation rates for the binding of IL-3 to the IL-3Ra are altered by truncation and by amino acid substitution at individual sites. Intracellular signaling studies using K116W and E43N demonstrate that differences in the IL-3 alpha binding characteristics are reflected in magnitude and kinetics of STAT5 phosphorylation.