Much of what is known of endothelial responses to cytokines has been derived from in vitro studies using cultured human umbilical vein endothelial cells (EC). Less is known of CNS EC responses and whether intact endothelium responds similarly to cultured cells. We have used techniques by which rat CNS microvessels can be isolated, then cultured in vitro, to study the response of intact endothelium to activation with cytokines. These microvessels are composed of viable EC and perivascular cells, predominantly pericytes. Expression of EC activation antigens in multicellular systems such as cultured microvessels can be assessed quantitatively using immunofluorescence laser cytometry. Interferon gamma increased immunologically reactive major histocompatibility complex class II antigens (< 300 to 2,398 +/- 225 average fluorescence intensity), while tumor necrosis factor alpha induced an increase in vascular cell adhesion molecule-1 (2,167 +/- 171) and E-selectin (1,628 +/- 315). CNS EC appeared to respond similarly to cultured EC with the exception that E-selectin expression was not transiently expressed but was maintained by microvessel EC for 24 and 48 h. Cultured CNS microvessels provide a good system for studying EC activation.