The myeloid-related proteins MRP-14 and MRP-8 and also p6, three calcium-binding proteins of the S100 family, translocate to the membrane during human neutrophil activation with stimuli known to require extracellular calcium for activity. When phorbol 12-myristate 13-acetate (PMA, an extracellular calcium-independent stimulus) is used, no translocation is observed. To characterize further the mechanisms involved in their translocation, phosphorylation of these proteins was studied. Three isoforms of MRP-14 were markedly phosphorylated in the membrane and in the cytosol upon activation with extracellular calcium-dependent stimuli, such as opsonized zymosan, the calcium ionophore A23187, N-formylmethionylleucylphenylalanine in the presence of cytochalasin B and arachidonic acid, or upon extracellular calcium-independent stimulation (PMA). In no case were p6 and a fourth, more basic isoform of MRP-14, phosphorylated. In PMA-activated cells, a phosphorylated acidic isoform of MRP-8 was detected in the cytosol only. However, phosphorylated MRP-8 represented only a small fraction of total MRP-8. Cgp 41251, an inhibitor of protein kinase C (PKC), completely inhibited the phosphorylation of MRP-8, and decreased cytosolic MRP-14 phosphorylation. To test whether phosphorylated MRP-8 could translocate, A23187, which induces translocation of the three S100 proteins, was added after PMA activation. This resulted in translocation of 18% +/- 5% of phosphorylated MRP-14 and 19% +/- 1% of only nonphosphorylated MRP-8. However, upon inhibition of PKC, translocation of MRP-14 and MRP-8 was increased up to 38% +/- 7% and 34% +/- 3% respectively. This suggests a putative role of phosphorylation and/or of PKC in the modulation of MRP-14 and MRP-8 translocation to the membrane.