Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor-vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-α and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca 2+, both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca 2+ increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-α, but not arachidonate, released Ca 2+ from endoplasmic stores and increased mitochondrial Ca 2+. Furthermore, Ca 2+ depletion abrogated TNF-α responses partially, but arachidonate responses completely. These differences in Ca 2+ mobilization by TNF-α and arachidonate were reflected in spatial patterning in the capillary in that the TNF-α effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-α-but not the arachidonate-induced responses. These findings indicate that the different modes of Ca 2+ mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-α revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.