The effect of a number of agents on the in vitro migration of guinea pig peritoneal macrophages and on the activity of macrophage migration inhibitory factor was studied. It was found that: 1. Dibutyryl cyclic adenosine monophosphate and theophylline cause pronounced inhibition of migration. Migration was not modified by cyclic guanosine monophosphate, noncyclic nucleotides or by prostaglandins E1, E2, and F2α. Theophylline (10-4M), paradoxically, abolishes the migration inhibitory effect of migration inhibitory factor. 2. Migration was not influenced by subtoxic doses of actinomycin D, puromycin and cycloheximide, but macrophages escaped inhibition partially, when exposed to migration inhibitory factor in the presence of puromycin (1-2 μg/ml) and of cycloheximide (0.1 μg/ml). 3. The polyanions dextran sulfate and heparin promoted macrophage migration and dextran sulfate (40 μg/ml) partially abolished migration inhibitory factor action. 4. Treatment of macrophages with the proteolytic enzymes trypsin and pronase resulted in enhanced migration while exposure to neuraminidase and wheat germ lipase was without effect. 5. Macrophage motility was dependent upon the presence of serum and increased in proportion with raising serum concentrations. Migration inhibitory factor was inactive at high serum concentrations. 6. Macrophage migration was enhanced and migration inhibitory factor activity abolished in the absence of calcium and magnesium.