Covalently linked pairs of cross-linked hemoglobin tetramers ("bis-tetramers", shown schematically as 6-8) were prepared by reacting hemoglobin A with tetrakis acyl phosphate esters (3-5). The effects of the link between tetramers are observed in the oxygen-binding properties of the bis-tetramers: they bind oxygen cooperatively but with Hill coefficients (n(50)) lower than that of the native protein and with a high average affinity. The bis-tetramers with longer connections between tetramers show a higher n(50), suggesting that steric interactions between the tetramers affect cooperativity. These results correlate to the observed reduced vasoactivity of heterogeneous solutions of oligomeric cross-linked hemoglobin tetramers.