In cells that do not express IC immunoglobulin light chain genes, the K enhancer-binding protein NF-KB is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-KB activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-KB can be found in the fraction from unstimulated 70213 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells. We conclude that both? OZ/3 and HeLa cells contain apparently cytosolic NF-KB in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.