The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP- and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrated the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs showed that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells.