Conjugates of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin (P-H) are promising photoactive agents for medical applications. As their ultimate efficacy will depend on the behavior of initial excited states, photophysical parameters have been determined with conventional steady-state absorption and fluorescence as well as time-resolved femto- and nanosecond spectroscopies. The fluorescence quantum yield of P-H and P-H conjugated to uncharged groups increases from approximately 0.03 in pH 7 buffer to approximately 0.05 in Triton X100 micelles (TX100) and in ethanol and to 0.12 in sodium dodecyl sulfate (SDS) micelles. Corresponding (1)S(1) lifetimes are approximately 5-10 ns. In buffer, an equilibrium between P-H monomers and small-size aggregates is observed. Conjugation with poly-S-lysine (P-(Lys)(n)) results in fluorescence quenching in all solvents. Structural reorganization of conjugates bearing a Di-O-isopropylidene-alpha-d-galactopyranosyl or a alpha/beta-d-galactopyranosyl group occurs in ethanol (k approximately 0.15 ps(-1)) after (1)S(1) state solvation (approximately 700 fs). Relaxation of bulky P-(Lys)(n) polypeptide chains takes place on a longer time scale in all solvents (k <or= 0.01 ps(-1)) with enhanced internal conversion. Triplet state ((3)T(1)) transient spectra of all derivatives in PBS, SDS, TX100, and ethanol exhibit a strong absorbance with a broad maximum in the 460-475 nm region and minor maxima at approximately 540, 630, and 690 nm. In ethanol, energy transfer from the P-H (3)T(1) state to beta-carotene provides an estimate of epsilon approximately 40,000 M(-1) cm(-1) at 460 nm for the P-H (3)T(1) state. Using triplet meso-tetraphenylporphyrin as an actinometer, the P-H triplet quantum yield (Phi(T)) is estimated to be approximately 0.50 in all solvents. This high Phi(T) leads to effective singlet oxygen production in buffered solutions.