Using the Boyden chamber invasion assay, the effect of 1 alpha,25-dihydroxyvitamin D3 [1 apha,25(OH)2D3] on the invasiveness of the highly invasive, oestrogen receptor-negative human breast cancer cell line MDA-MB-231 was examined. The MDA-MB-231 cells were shown to contain high-affinity receptors for 1 alpha,25(OH)2D3 with a Kd of 1.5 x 10(-11) M. When the cells were treated with 1 alpha,25(OH)2D3 for 4 days before the assay was performed, a dose-dependent inhibition of their invasive potential was demonstrated. Fifty per cent inhibition of invasion was obtained with a concentration of 13 pM of 1 alpha,25(OH)2D3. However, when the cells were treated for only 6 h during the assay, no inhibitory effect was seen. The process of migration was also affected by treatment with 1 alpha,25(OH)2D3 for 4 days, although the inhibition was not of the same magnitude as seen for the invasion. Fifty per cent inhibition of migration occurred at a concentration of 3.2 nM of 1 alpha,25(OH)2D3 (250 times higher than in the invasion assay). Inhibition of invasion and migration was not due to the known anti-proliferative effect of 1 alpha,25(OH)2D3, as no growth reduction could be demonstrated with treatment up to 5 days. Based on the present investigation it can therefore be concluded that 1 alpha,25(OH)2D3 is able to inhibit tumour cell invasiveness by a mechanism which is not exclusively based on its anti-proliferative and anti-migrative effects.