Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. Mutations in this gene cause the hereditary stroke disorder CADASIL, which is associated with degeneration of peripheral vascular smooth muscle cells in several vascular beds, indicating important although unknown vascular functions of notch3. Recently, severe renal injury was found in CADASIL patients without co-exciting nephropathy, indicating a role of notch3 in renal microcirculation. To test if the ANG II signalling cascade was affected in notch3-/-, we explored contractility and Ca2+i signalling in the afferent arteriole (AA). Notch3/-AAs were unable to maintain ANG II-induce d contractions longer than 2 min compared to +/+ littermates (relative diameter: 0.63±0.06 vs. 0.92±0.05, respectively, p< 0.05, t=6 min), indicating reduced contractile endurance. There was no difference in contractility if external Ca2+ was removed from the medium, however, indicating compromised Ca2+ entry. The ANG II Ca2+i response in fura-2 loaded AAs was reduced in notch3-/-compared to + /+ (340/380 fura-2 ratio-change: 0.38±0.04 vs. 0.17±0.03, respectively, p< 0.01). This difference disappeared with Ca2+ removal or nifedipine blockade of the L-type Ca2+ channel. If activated directly by KCl-depolarization, however, L-type Ca2+ entry was similar between the strains. Screening the expression of 45 contractility-related genes revealed many-fold down-regulation of the T-type Ca2+ channel Cav3.2 in notch3-/-. The Cav3.2 blocker mibefradil markedly reduced the ANG II Ca2+i response notch3+/+ (0.15±0.09, p< 0.05), but had no effect in -/(-0.04±0.03, NS). In conclusion, reduced contractile endurance in notch3-/-AAs is caused by an un-coupling of ANG II stimulation and Ca2+-entry, likely caused by compromised function of the Cav3.2. To our knowledge, this is the first time dys-regulation of a specific contractile protein has been linked to noth3 expression and vascular function.