Chemotaxis is one of the most important functions of the polymorphonuclear leukocyte (PMN). In the host defense against pyelonephritis, the renal medulla is a site of interaction between bacteria and PMNs. At this site the osmotic pressure is elevated due to a high concentration of NaCl and urea. We evaluated the in vitro chemotactic activity of PMNs under the hyperosmolar conditions created by high concentrations of NaCl and urea. This activity was suppressed by the stimulation of opsonized zymosan and formyl-methionyl-leucyl-phenylalanine. The inhibition of chemotaxis was partially preserved by phosphoenolpyruvic acid (PEP), a precursor of adenosine triphosphate (ATP), in hyperosmolar NaCl but not in urea. The intracellular content of ATP was increased by supplementing the hyperosmolar NaCl with PEP. These observations suggest that inhibition of the chemotactic activity of PMNs is due to differing mechanisms for each NaCl and urea, and that PEP may protect the PMNs against hyperosmolar NaCl by maintaining ATP content.