This paper presents data which allow one to characterize pyridoxal 5 -phosphate (PLP) as an optical label for electrostatic potential measurements in proteins. Experimental studies were carried out with 6-N-(5 -phosphopyridoxyl)-2-N-acetyl-lysyl methyl ester (PLP-ALME) as a model compound which simulates PLP covalently bound to a protein. Calculations of electrostatic potential maps were done using the model compound 2,4-dimethyl-3-hydroxy-5-hydroxymethyl phosphate-pyridine (DHHPP). Studies on relative changes in PLP-ALME fluorescence and absorbance vs. pH resulted in four pK a values of PLP-ALME in solution which can be used as intrinsic pK a values (pK i n t ) of the ionizable groups of the label covalently bound to the protein. The pK a values obtained from fluorescence data are 4.1, 5.6, 8.7 and 11.1 and those from absorbance data are 3.1, 4.7, 8.7 and 11.0. The differences between corresponding pK a values are related to differences in PLP-ALME behaviour between the excited and ground electronic states and to intramolecular charge-charge interactions. Quenching of PLP-ALME fluorescence by I - , acrylamide and Cs + at pH 6, 7 and 8 shows that in the case of I - and acrylamide the Stern-Volmer constants of quenching (K S V ) decrease with increasing pH, while the opposite is true for K S V of Cs + . These results as well as the analysis of electrostatic potential maps of DHHPP show that with its ampholytic character PLP can be used to measure local electrostatic potentials in the pH range 5-9.