The recent introduction of fluorescent two-dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30-day-old kittens, in order to identify proteins expressed in an age-related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two-dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.