Background and Purpose—Previous studies have shown that contractile endothelin type B (ETB) receptors are upregulated in cerebral arteries after experimental focal cerebral ischemia. The aim of this study was to examine the upregulation of contractile ETBreceptors in cerebral arteries after organ culture and to elucidate the intracellular pathways involved.Methods—Rat middle cerebral arteries (MCAs) were incubated with or without inhibitors. The vessels were mounted in myographs, and the contractile responses to endothelin-1 (ET-1) (ETAand ETBreceptor agonist) and sarafotoxin 6c (ETBreceptor agonist) were measured. Levels of ETBreceptor mRNA were measured with real-time polymerase chain reaction.Results—In fresh MCA, sarafotoxin 6c had no contractile effect. However, after organ culture, a strong concentration-dependent contraction was induced. ET-1 produced a strong contraction, in which the Emaxwas unaffected by organ culture but the EC50was decreased with time. The sarafotoxin 6c–induced contraction after 24 hours of organ culture was attenuated by the transcriptional inhibitor actinomycin D and the translational inhibitor cycloheximide as well as the protein kinase C inhibitor Ro-31-8220. Real-time polymerase chain reaction revealed that the mRNA levels of the ETBreceptor were increased after organ culture compared with fresh vessels. Actinomycin D and Ro-31-8220 diminished the enhanced mRNA levels considerably.Conclusions—The results suggest that, in fresh MCA, the ETAreceptor is the most prominent subtype, while after organ culture ETBreceptors also contribute to the contraction. This upregulation is due to de novo transcription of receptors. Protein kinase C is involved in the upregulation as Ro-31-8220 attenuates the contraction and the mRNA increase.