Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca2+ signaling in ear arterioles. Conscious mice were used in order to preserve normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca2+ indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s−1. In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5–6 μm magnitude was accompanied by nearly uniform [Ca2+] oscillations from ~0.1 to 0.5 μM, with maximum [Ca2+] occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform “Ca2+ flashes” were also observed but not asynchronous propagating Ca2+ waves. In contrast, vasomotion and dynamic Ca2+ signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium (30 μg/g BW, i.p.) caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca2+ transients.