To gain insight into the mechanisms involved in regulating murine interleukin-3 (mIL-3) receptor expression, we have examined the effects of mIL-3 and murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) on mIL-3 receptor internalization and re-expression and studied the relationship between mIL-3 cell surface receptor density and growth factor sensitivity. As a source of cells for our studies, we used a B6SUtA clone, B6SUtA1, which grows equally well in mIL-3 or mGM- CSF when supplemented with 20% fetal calf serum (FCS) in RPMI 1640. Intracellular processing studies carried out in the presence and absence of methylamine suggested that mIL-3 is cleaved at two specific sites before its complete digestion within lysosomes. However, unlike its ligand, cycloheximide studies indicated that internalized mIL-3 receptors are recycled to the cell surface. When B6SUtA1 cells were continuously passaged in mIL-3, cell populations allowed to exhaust the mIL-3 in the medium (high density cells) expressed more than ten times (ie, approximately 100,000/cell) the mIL-3 receptor number of those growing exponentially at low cell concentrations (low density cells). Since the high density cells were no larger than the low density cells, the marked increase in mIL-3 receptor number per cell reflects a true up-regulation of receptor expression. A kinetic analysis of this up- regulation revealed that it begins within one hour of mIL-3 exhaustion. Moreover, proliferation assays with these two cell populations, using 3H-thymidine incorporation, suggested that the high density cells were 30-fold more responsive to mIL-3. However, when B6SUtA1 cells were passaged in mGM-CSF, there was no difference in mIL-3 receptor number between high density and low density cells (ie, approximately 100,000/cell). Identical studies carried out with another mIL-3 dependent cell line, 32D C3, demonstrated that this phenomenon was not unique to B6SUtA1 cells.