Volume changes associated with CO photodissociation from fully reduced cytochrome aa 3 have been determined using photoacoustic calorimetry (PAC). Analysis of PAC data reveals two intermediate steps in the photodissociation reaction with volume changes of +6.8 and +6.9 mL/mol occurring on time scales of <50 ns and ∼1.5 μs, respectively (T = 22 °C). The value of ΔV for the fast process can be attributed to conformational dynamics associated with photoinduced transfer of CO from the high-potential heme a 3 to the high-potential CuB site, while the slower phase reaction occurs on a time scale consistent with the thermal dissociation of CO from CuB. It is speculated that these volume changes may be linked to movement of Glu-242 as part of a proton shuttle mechanism in heme/copper oxidases.