Previously we have reported that Ca2+ concentration in culture medium affects the morphogene-sis of HVJ from infected cells. We further investigated the effects of Ca2+ on HVJ morphogenesis in LLC-MK2 cells, focusing on M (matrix) protein which plays an important role in the assembly of viral components. On culture in standard medium (containing 1.8 mM Ca2+), most of the M protein accumulated on the apical surface in patches. On double immunofluorescent staining with anti-M protein monoclonal antibody and polyclonal antibody against HVJ glycoproteins, M protein and viral glycoproteins were observed in the same patches at the cell surface. By contrast, in low Ca2+ medium, M protein was evenly dispersed over the cell surface, especially in the regions of cell-cell contact, and was not seen in patches. Immunoblot analysis for M protein in the infected cells showed that the total amount of M protein was almost the same irrespective of Ca2+ concentration in the culture medium. But, there was a distinct difference in the forms of M protein found in the cells: most of the M protein in the cells cultured in standard medium was of the non-phosphorylated form, whereas the phosphorylated form of M protein was distinctly increased in the cells cultured in low Ca2+ medium. These results suggest that Ca2+-deficiency in the medium blocks dephosphorylation of phosphorylated M protein necessary for patch formation, resulting in disturbance of viral morphogenesis. © 1994, Japan Society for Cell Biology. All rights reserved.