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Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated P(NPC3):GUS and P(NPC4):GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. P(NPC4):GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 μM BL. BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 μM BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.