L-cell colony-stimulating factor (CSF) is identical to macrophage growth factor and stimulates macrophage proliferation (Stanley et al., 1976, J. Exp. Med. 143: 631-647). The nature of the interaction of iodinated L-cell CSF (125I-CSF) with murine peritoneal exudate macrophages was studied. On incubation with 10 pM 125I-CSF at 0 degrees C, cellular binding of 125I-CSF reaches a stable maximum within 15 h. This is in contrast to the association behavior at higher temperatures. At 37 degrees C, cell-associated 125I-CSF levels reach, within 45 min, an unstable maximum which is up to 10-fold less than that occurring under the same conditions at 0 degrees C. At 0 degrees C, binding is saturated (approximately 5 X 10(4) sites/cell) at CSF concentrations of 1 nM. A comparison of binding and competition experiments indicates that iodinated L-cell CSF binds as effectively as L-cell CSF and that human urinary CSF and L-cell CSF equipotently compete for 125I-CSF binding. Specificity of the CSF-binding site is demonstrated by the failure of other known growth factors and hormones to compete for 125I-CSF binding. These studies and other findings suggest that 125I-CSF binding is restricted to macrophages and their precursors and to macrophage cell lines and that the binding site(s) is the receptor mediating the biological action of this CSF.