μ-Calpain is a calcium-dependent neutral thiol protease activated by micromolar concentrations of calcium. μ-Calpain is implicated in various cellular functions regulated by calcium including exocytosis, cell fusion, apoptosis and control of cell proliferation. We studied the effect of 1,25-(OH)2D3 on μ-calpain levels in the human renal cell carcinoma line SK-RC-29 using monoclonal antibodies to the 80 kDa subunit of μ-calpain. Exposure of low density cultures (15000 cells/cm2) to 1,25-(OH)2D3 (100nM) for 48 hours resulted in 1.5-3 fold increase of μ-calpain cell content. The effect was not observed in higher density cultures (40000 cells/cm2). μ-Calpain content of high density cultures was higher than that of low density cultures and similar to that in low density cultures treated by 1,25-(OH)2D3. The cellular content of two other calcium binding proteins, annexin II and annexin VI was not affected by the hormone. 1,25-(OH)2D3 did not affect cell number or variability therefore its effect on μ-calpain is not secondary to changes in cell density. The effect of 1,25-(OH)2D3 was dose-dependent apparent already at 1nM and was not observed with 24,25-(OH)2D3. Increase in μ-calpain content may underlie some of the actions of 1,25-(OH)2D3 on classical and non classical target cells.