We suggest a method of quantitating the motile actions of surface protrusions, in spreading animal cells in culture. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. Studying 3T3 cells with this assay, we found that the presence of Na +, K +, Cl-, and Mg ++ or Ca ++ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the cells for at least 50 min.Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of three glucose analogues which are generally believed to be nonmetabolizable cannot restore the activity.Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Furthermore, serum codetermines which of the major surface extensions, filopodia, lamellipodia, or lobopodia, is predominantly active.We found three distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B of the particle removal are very similar in all three cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal threefold.As long as we do not fully understand the logic and mechanisms underlying the phenomena of animal cell motility, they will continue to appear polymorphous and erratic. A necessary first step toward such understanding is the development of quantitative assays for the phenomena of animal cell motility. The locomotion, i.e., the displacement of whole cells on an artificial substrate, has been successfully quantitated (12,17), as have the translocational and rotational movements of single molecules within the plasma membrane (13,22,26), the collective movement of many antigenic sites on the cell surface, i.e., the capping phenomenon (16,34), and the movement of single, sur-