Diffusion processes of CO2 into or out of a thin layer of myoglobin (Mb) solution were followed by pH-sensitive fluorescence of 4-methylumbelliferone. Mb solutions were prepared by dissolving horse heart Mb at 0.1 to 4 mM in a modified Krebs solution containing NaHCO3 of 30 mM. Carbonic anhydrase was added to observe the diffusion-limited pH changes. The PCO2 in the layer were calculated as the numerical solution of a diffusion equation. For the simulation of the observed pH-time curves, the PCO2 changes were converted to the pH changes using a linear relation between logPCO2 and pH. The diffusion coefficients of CO2 and HCO3- (DCO2 and DHCO3) were determined as the optimum parameters to fit the calculated pH-time curves to the observed ones. Both the DCO2 and DHCO3 decreased exponentially as the Mb concentration was increased. At a physiological concentration of Mb in cardiomyocytes (0.2 mM) and at 37 degrees C, the DCO2 and DHCO3 values were 9.8 x 10(-5) and 16 x 10(-5) cm2.s-1, respectively. The buffer value (beta) was calculated as the slope of a pH-bicarbonate diagram by measuring the CO2 content and pH of the Mb solutions equilibrated with known PCO2 gases. The beta was found to increase with increasing Mb concentration with a value of 6.2 mEq.l-1.pH-1 at 0.2 mM.