Metabolically radiolabeled recombinant proteins were used to systematically evaluate peptide recoveries from in-gel trypsin digestion.At least 80% of the labeled tryptic peptides could be readily extracted from gel bands containing 1 to 10 pmol, and at least 70% could be extracted at 200- to 500-fmol levels using a recombinant 52-kd protein. Alkylation before electrophoresis or before trypsin digestion had minimal effects on peptide recovery; although alkylation, especially before gel analysis,may reduce heterogeneity of resulting peptides containing cysteines. Comparison of different gel thicknesses using unminced gel bands suggested that 1.0-mm gels were optimal. Surprisingly, peptide recoveries from 0.5-mm gels were low and variable, primarily because of increased diffusion of protein out of thin gels during fixing and staining. Although 70% to 85% of tryptic peptides could typically be extracted from gels over a range of conditions and protein concentrations, further processing of peptide extracts resulted in substantial additional losses. Even minimal handling resulted in loss of about 10% to 15% of extracted peptides by adsorption to plastic surfaces. Adsorptive losses were particularly high, sometimes exceeding 50%, and variable if extracts were partially dried in a Speedvac to concentrate the sample or to remove acetonitrile. High acetonitrile extraction and/or Speedvac concentration appear to be detrimental, and their elimination simplifies sample handling and automation. SYPRO Ruby Red, a sensitive noncovalent fluorescent stain appears to be an attractive alternative to Coomassie blue for in-gel trypsin digestion. These results suggest an optimized in-gel trypsin digestion strategy in which proteins in 1.0-mm-thick gels are stained with Coomassie blue or Ruby Red, digested overnight with modified trypsin, and extracted one or two times with small volumes of aqueous buffer. It is especially critical that subsequent surface exposure be minimized, and concentration by vacuum drying should be avoided.