Alpha hemoglobin stabilizing protein (AHSP) reversibly binds nascent alpha globin to maintain its native structure and facilitate its incorporation into hemoglobin A. Previous studies indicate that some naturally occurring human alpha globin mutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect hemoglobin A production through AHSP-independent effects, including reduced binding to beta globin. We analyzed 6 human alpha globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S, and P119S impaired interactions with both AHSP and beta globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N alpha globins bind beta globin normally but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In Escherichia coli and erythroid cells, alpha globin K99E stability is rescued on coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some alpha globin variants and support the hypothesis that AHSP promotes alpha globin chain stability during human erythropoiesis.