Consumption of the intercellular messenger, nitric oxide (NO), by extracellular hemoglobin causes the hypertensive side effect associated with current blood substitute products. Substitution of large aromatic amino acid side chains into the distal cavity of hemoglobin can decrease the rate of this NO scavenging reaction, and mitigate the hypertensive effect. However, such substitutions can also affect O2 binding and release by hemoglobin. To better define the impact of distal pocket mutation on O2 transport, several myoglobin prototypes were tested in an artificial capillary. In addition, a series of recombinant hemoglobins containing phenylalanine and tryptophan substitutions at key locations were characterized to examine how these mutations affect O2 and CO binding. Experiments measuring O2 release and uptake by mutant myoglobins in an artificial capillary demonstrate that delivery is dependent on the affinity of the protein, but that uptake is similar for all of the mutants. This shows that decreases in association rate constants are better tolerated than decreases in dissociation rate constants, and validates the connection between the equilibrium constant for O2 binding, and the physiological transport function of hemoglobin. Placement of Phe and Trp at positions B10, E11 and G8 within the distal pocket decreases the rate at which ligands can gain entry to the active site, which was expected based on similar behavior in myoglobin mutants. The B10 mutants have direct steric and electrostatic interactions with the ligand, seen in the 2000-fold decrease in rate of O2 association for the beta(Trp(B10)) and 60-fold decrease in the O2 dissociation rate constant for alpha(Phe(B10)) subunits. Substitution at position E11 increases O2 and CO affinity due to removal of the naturally occurring gamma2CH3 group of Val(E 11). The E11 mutants have less dramatic effects in hemoglobin than in myoglobin due to the absence of 'extra' volume in the back of the distal cavity in either hemoglobin subunit. Steric crowding by position G8 mutants decreases the rate and extent of ligand capture, particularly in beta subunits. In general, ideas developed for ligand binding in myoglobin translate well to hemoglobin, but structural details of each subunit can magnify or diminish the effect of the mutation.