The application of mononuclear cell populations to a nylon wool (NW) column is a common early procedure in the selection of T lymphocytes for further study. The technique as presently employed in various laboratories does not appear to be standardized, and surprisingly little is known about the effects of physicochemical alterations and biologic mediators on the interaction of lymphocytes with the NW substrate. In this study the factors controlling the adherence of rat splenocytes to NW were examined. NW adherence was shown to be independent of loaded cell concentration, column packing or pH, but was very dependent on NW column size, wash volume, incubation time and ambient temperature. The addition of protein to media did not alter lymphocyte NW adherence, and the interaction did not appear to depend on intact cellular glycolysis or protein synthesis or on microtubular or microfilament function. Because of the theoretical importance of surface adherence to cell motility, the effects of various agents that alter lymphocyte migration were tested in the lymphocyte NW assay. Of the positive chemokinetic factors tested, only casein altered (decreased) NW adherence. Of the negative chemokinetic principles tested only the human lymphokine LyMIF altered (increased) NW adherence. The studies show that the NW-nonadherent cell pool may be a heterogeneous population depending on the physical conditions of the assay, and the NW adherence of rat splenocytes is not an all-or-none phenomenon but can be altered by physical and biological factors. This makes the standardization of the assay of critical importance, particularly if one wishes to compare results of subsequent experiments between laboratories.