We have produced recombinant hemoglobins (rHbs) alpha 2 beta 2(6Glu-- >Lys) (rHb beta E6K) and alpha 2 beta 2(6Glu-->Arg) (rHb beta E6R) using a yeast expression system coupled with a polymerase chain reaction (PCR)-based mutagenesis strategy for studies focused on defining determinants that facilitate crystallization of Hb C (alpha 2 beta 2(6Lys)). rHb beta E6K had the same electrophoretic mobility as native human Hb C, whereas rHb beta E6R migrated slightly slower than Hb C on cellulose acetate electrophoresis. The carbonmonoxy (CO) forms of rHb beta E6K and rHb beta E6R formed tetrahedral crystals in vitro in 2.3 mol/L phosphate buffer just like native Hb C. The Hb concentration required for crystallization of CO-rHb beta E6R was lower than that of CO-rHb beta E6K, suggesting that stronger basic amino acids at the beta 6 position accelerate crystallization of Hb. However, the size of rHb beta E6R crystals was smaller than that of rHb beta E6K. Crystallization of native Hb C and both rHbs was inhibited by Hb F. These results suggest that alpha 2 beta gamma-heterohybrids that have basic amino acids at the beta 6 position behave similarly and are unable to crystallize like Hb C.