We previously reported that multinucleated macrophages express a high concentration of Na(+)-K(+)-ATPases that are concentrated on the nonadherent domain of their plasma membrane (A. Vignery, T. Niven-Fairchild, D. H. Ingbar, and M. Caplan. J. Histochem. Cytochem. 37: 1265-1271, 1989). We also showed that an increase in newly synthesized alpha-subunit occurred during cell culture and multinucleation. We now present evidence that macrophage multinucleation in vitro is accompanied by an increased accumulation of Na(+)-K(+)-ATPase alpha-subunit mRNA. Most interesting is the detection of significant amount of both alpha 1- and alpha 3-isoform mRNA and peptide in these cells by in situ hybridization, Northern and Western blot analyses. These qualitative and quantitative variations in Na(+)-K(+)-ATPase expression suggest that macrophage multinucleation is accompanied by a coordinated regulation of gene expression and that multinucleation confers a specific function to macrophages. Multinucleated macrophages offer a novel model system to investigate not only the specific function(s) of the alpha 3-isoform but also the role of the Na(+)-K(+)-ATPase in giant cells and osteoclasts.