Photoacoustic calorimetry and steady-state spectroscopic techniques are used to investigate the wavelength-dependent photoreactivity of trans-urocanic acid under representative physiological conditions in vitro. The maxima of the emission and excitation spectra and the value of Delta(r)H, the reaction enthalpy as determined from photoacoustic measurements, varied as a function of the excitation wavelength. From these data, we are able to confirm that the photolysis of trans-UA at 266 nm (the peak of the absorption spectrum where isomerization is inefficient) generates a long-lived electronically excited tripler state that lies approximately 230 kJ/mol above the ground state. Isomerization does not occur from this triplet electronic state, but bimolecular energy transfer to O-2, generating (1) Delta(g) (O-2), does occur. Excitation of trans-UA at 308 nm (in the tail of the absorption spectrum) does not lead to triplet state formation. Instead, excitation at this wavelength leads to isomerization. Using measured isomerization quantum yields at this photolysis wavelength (Phi = 0.49), we are able to determine that the ground state of cis-UA lies about 40 kJ/mol above that of the trans isomer. Photoacoustic data recorded following the photolysis of cis-UA at 266 and 308 nm show the same trends, supporting the conclusion that the wavelength-dependent chemistry is not due to the presence of multiple ground-state rotamers. The data indicate that the broad structureless absorption spectrum of trans-UA is comprised of overlapping transitions due to at least two distinct electronic states. The different reactivities of these two states result in the wavelength-dependent isomerization yields that have been measured for trans-UA in the UV-B (280-320 nm) range.