Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently been shown to act as a second messenger controlling intracellular Ca²⁺ responses in mammalian cells. Many questions remain regarding this signaling pathway, including the role of the ryanodine receptor (RyR) in NAADP-induced Ca²⁺ transients. Furthermore, the exact metabolic pathway responsible for the synthesis of NAADP in vivo has not been determined. Here, we demonstrate that the NAADP mediated Ca²⁺ release system is present in human myometrial cells. We also demonstrate that human myometrial cells use the NAADP second messenger system to generate intracellular Ca²⁺ transients in response to histamine. It has been proposed in the past that the NAADP system in mammalian cells is dependent on the presence of functional RyRs. Here, we observed that the histamine-induced Ca²⁺ transients are dependent on both the NAADP and inositol 1,4,5-trisphosphate signaling pathways but are independent of RyRs. The enzyme CD38 has been shown to catalyze the synthesis of NAADP in vitro by the base-exchange reaction. Furthermore, it has been proposed that this enzyme is responsible for the intracellular generation of NAADP in vivo. Using CD38 knockout mice, we observed that both the basal and histamine stimulated levels of NAADP are independent of CD38 and the base-exchange reaction. Our group is the first to demonstrate that NAADP is a second messenger for histamine-elicited Ca²⁺ transients in human myometrial cells. Furthermore, the NAADP mediated mechanism in mammalian cells can be independent of RyRs and CD38. Our data provides novel insights into the understanding of the mechanism of action and metabolism of this new second messenger system. Copyright © 2007 the American Physiological Society.