1. A dual-wavelength microfluorimetric method using Fura-2 as calcium indicator was applied to cells from an immortalized cell line of rat brain microvascular endothelial cells (RBE4), and to primary cultured rat brain endothelial cells. 2. In RBE4 cells, a brief (20 s) pulse of extracellular ATP (100 microM) induced a transient increase in the cytoplasmic calcium level ([Ca2+]i). Control responses to 100 microM ATP consisted of a ratio increase of 0.64 +/- 0.03 (mean +/- s.e., n = 51). Responses were seen at a concentration of 2.5 microM and were maximal at 100-1000 microM. When extracellular calcium was chelated with EGTA, the transient increase in [Ca2+]i was not affected. The results are consistent with Ca2+ mobilization from intracellular stores. 3. The purinoceptor involved belongs to the P2 subtype, since the agonist potency order among the adenine nucleotides was ATP > ADP > AMP. Moreover, the increase in [Ca2+]i evoked by ATP was partially inhibited by the P2 antagonist, suramin but was not affected by 8-phenyltheophylline, a P1-purinoceptor antagonist. The strong desensitization observed with repeated applications of ATP is also typical of a P2 receptor. 4. 2-Methylthio-ATP (2meS-ATP 100 microM), a P2Y agonist, elevated [Ca2+]i in only 17% of the cells tested; however, 2meS-ATP was found to antagonize the effect of ATP in all cells tested. The increase in [Ca2+]i evoked by ATP was inhibited by 500 s application of the P2Y purinoceptor antagonist, Reactive Blue 2 at 10 microM, while 60 s application of 100 microM was ineffective. 5. The uracil nucleotide, UTP (100 microM) was as effective as ATP in increasing [Ca2+]i. The effects of ATP and UTP were not additive. Cells desensitized to the action of ATP (or UTP) were unable to respond to UTP (or ATP).6. alpha,beta Methylene-ATP (alpha,beta meATP 100 microM), a P2x, agonist, elevated [Ca2+], in only 40% of the cells tested. In these cells it was less effective than ATP in increasing [Ca2+]i.7. Cells desensitized to the action of ADP responded, to a smaller extent, to ATP. In contrast, cells desensitized to the action of ATP were unable to respond to ADP.8. On primary cultures of brain endothelial cells the increase in [Ca2+]i in response to extracellular ATP(100 microM) and UTP (100 microM) was of an equivalent amplitude, and similar to the response in RBE4 cells.The pattern of desensitization was also similar to that in RBE4 cells.9 This comparative study indicates that in well-characterized brain microvascular endothelial cells that retain brain endothelial characteristics, the major class of nucleotide receptor is of the P2mu type. The implications for physiology are discussed.