The tissue factor (TF)-initiated coagulation pathway plays important roles in hemostasis, inflammation, metastasis, and angiogenesis. Phosphorylation of the TF cytoplasmic domain is functionally relevant in metastasis. How TF cytoplasmic domain phosphorylation downstream of protein kinase C (PKC) activation is regulated in primary vascular cells remains poorly understood. Here, phosphorylation of Ser258, rather than the PKC consensus site Ser253, is identified as the major conformational switch required for recognition by a phosphorylation-specific antibody. With this novel reagent, we demonstrate that the TF cytoplasmic domain is primarily unphosphorylated in confluent endothelial cells. TF cytoplasmic domain phosphorylation can occur in the absence of the autologous TF transmembrane and extracellular domains but requires maturation of TF in the Golgi compartment and cell surface expression. Site-directed mutagenesis and 2-bromopalmitate treatment provide evidence that palmitoylation of the cytoplasmic Cys245 is a negative regulatory mechanism of Ser258 phosphorylation. Profiling with PKC-selective inhibitors identifies PKCalpha as important for TF cytoplasmic domain phosphorylation. Mutagenesis of protein kinase consensus sites are consistent with a model in which PKC-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a Pro-directed kinase. Thus, cell surface location-dependent phosphorylation of the TF cytoplasmic domain is regulated at multiple levels.