Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY FI C1-IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on p/'s of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (Thio-Glo I and Rhodamine Red C2 maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY FI C1-IA or Rhodamine Red C2 maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification.