X-ray absorption fine structures of photolyzed carboxymyoglobin were measured at 4 K and at 40 K. The latter was a 10-s-resolved measurement following photolysis. The goal of the project was to elucidate the dynamic pathways through which ligand binding couples to protein conformation changes. The project involved five major tasks: (1) We constructed an integration spectrometer for x-ray absorption capable of 100 μs-resolved measurement. (2) Heme protein samples have to be photolyzable on the one hand and possess a high (Fe) fluorescence yield on the other. This is very difficult to achieve with solution samples. The problem was solved by embedding concentrated (∼10 mM) myoglobin in thin (∼20 μm) dry polyvinyl alcohol (PVA) films. Carboxymyoglobin embedded in PVA film, has the same properties as in frozen buffer solution as proven by optical absorption, kinetics of CO recombination following photolysis and x-ray absorption spectra. (3) A cryostat was constructed for x-ray absorption measurement, which was also equipped with optical windows for flash photolysis and optical monitoring of the degree of photodissociation in the sample. (4) Synchrotron radiation experiments were performed at Stanford Synchrotron Radiation Laboratory and at National Synchrotron Light Source at Brookhaven. (5) Data analyses showed that the photolyzed carboxymyoglobin (MB\sp∗) is structurally different from either carboxymyoglobin (MbCO) or deoxymyoglobin (Mb): the Fe-C(O) distance is 1.9 A, 2.17 A, ∞ A for MbCO, Mb\sp∗ and Mb respectively; the corresponding Fe-N\sbimidazole distances are 2.2 A, 2.18 A, 2.1 A and Fe-N\sbporphyrin distances are 1.97 A, 1.98 A, 2.06 A. At 40 K, from 1 s to 10 s following photolysis, 40% of photodissociated CO has recombined with Fe, and the rest of the sample is indistinguishable from Mb\sp∗. The structure of Mb\sp∗ described above is in disagreement with a previous report by Powers et al. (Biochemistry 23, 5519 (1984)).