In the migration under agarose assay for measurement of polymorphonuclear leukocyte chemotaxis and random migration, the agarose gel ordinarily contains 10% human serum or plasma. Influences of serum and plasma on cell movement using this assay were defined. Utilizing plasma as protein source allowed more random migration and chemotaxis than when serum was used. Heat inactivation of both serum and plasma decreased random migration and chemotaxis. If heating at 56°C was prolonged beyond 15-30 min, leukocyte movement was augmented. Excessive concentrations of platelets or platelet products, or heparin in agarose, also augmented migration, but storage of plasma or sera at various temperatures for 24 h did not appreciably affect migration. Large amounts of hemoglobin in agarose decreased chemotaxis, but the effect of trace amounts of hemolysis was negligible. Handling plasma or sera in standard ways prior to use in the under agarose assay may make this assay more reproducible and sensitive.