We have reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] induces fusion of mouse alveolar macrophages directly by a mechanism involving spermidine-dependent protein synthesis (Tanaka, H. et. al., 1989, Exp. Cell Res. 180, 72-83). The macrophage fusion induced by 1 alpha,25(OH)2D3 occurred in a calcium-dependent manner (Jin, C.H. et al., 1988, J. Cell. Physiol. 137, 110-116). In the present study, we examined the possibility that transglutaminase, a calcium-dependent enzyme, is involved in the fusion of macrophages induced by 1 alpha,25(OH)2D3. The activity of transglutaminase increased greatly 12 h after 1 alpha,25(OH)2D3 was ended and reached a maximum at 48 h. Western blot analysis of the cell lysate using an anti-transglutaminase antibody showed that 1 alpha,25(OH)2D3 induced a 77-kDa protein corresponding to transglutaminase. When spermidine synthesis was inhibited by adding methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, the increase in the transglutaminase synthesis by 1 alpha,25(OH)2D3 was markedly inhibited with concomitant inhibition of fusion. Adding more spermidine restored both the synthesis of transglutaminase and the fusion. The treatment of macrophages with cystamine, an inhibitor of transglutaminase, inhibited the fusion in parallel with the suppression of transglutaminase activity, both induced by 1 alpha,25(OH)2D3. These results clearly indicate that 1 alpha,25(OH)2D3 induces transglutaminase by a spermidine-dependent mechanism and that this enzyme is involved in a biological reaction(s) essential for inducing macrophage fusion.