Sorting of fluorescent cells is a powerful technique for revealing the cellular processes that differ among the various cell types found in complex tissues. With the recent availability of transgenic mouse strains in which specific subpopulations of neurons are labeled, it has become desirable to purify these fluorescent neurons from their surrounding hetereogeneous brain tissue for electrophysiological, biochemical and molecular analyses. This has been accomplished using automated fluorescence-activated cell sorting (FACS) and laser capture microdissection (LCM). Although these procedures can be effective, they have some serious disadvantages, including high equipment costs and difficulty in obtaining samples completely free of contaminating tissue. Here we offer an alternative protocol for purifying fluorescent neurons, which relies on less-expensive equipment, readily produces perfectly pure samples and can be applied to neurons that are only dimly labeled and present in low numbers. The entire protocol can be completed in 3-5 h.