X-ray crystallographic analyses of myoglobin (Mb) in the deoxy and GO-bound states noted the absence of a preformed pathway for ligand movement from the heme iron to the solvent. To explore a mechanism of ligand entry, time-resolved UV resonance Raman experiments have been carried out, using a mutant with tyrosine at the distal histidine position. The results indicate the presence of a transient, open pathway which is generated after photodissociation of CO in the H64Y mutant. The Raman spectra were probed at 235 and 416 nm with a time resolution of 7 ns in the range -100 ns to 10 ms following photolysis at 532 nm. In the 235 nm excited spectra, the tyrosine bands of H64Y Mb at 1618 (Y8a) and 1176 cm(-1) (Y9a) are noticeably intensity-enhanced shortly after photolysis, but the original intensity is restored by 5 ms. The time range in which the Tyr bands are intensified is prolonged by addition of glycerol to the solvent. This intensity change is not seen with the native Mb which has three naturally occurring Tyr residues. Thus, the intensity increase observed for the mutant Mb is attributed to Tyr64. The corresponding bands of the p-cresol derivative exhibit greater intensity in polar (H-bond-forming) solvents than in nonpolar solvents. This result suggests that the increase in intensity of the Tyr bands in the transient form of the mutant myoglobin compared to that in the equilibrium form is due to exposure of Tyr64 to solvent water. Increased solvation indicates an outward movement of the phenol side chain and formation of an open channel to the distal pocket.