Anoikis is a specific type of apoptosis caused by the detachment of anchorage-dependent cells from their supportive matrix. Aggregation of suspended cells is believed to suppress anoikis. Here we describe the effects of cellular aggregation on anoikis in Madin-Darby canine kidney (MDCK) cells. Suspension cultures of MDCK cells grown under conditions known to induce extensive cellular aggregation were less able to reattach to culture dishes, exhibited higher caspase-8 activity, and contained more sub-G(1) cells than suspension cultures did with less cellular aggregation. When suspension cultures of MDCK cells were separated into aggregated cells and single cells, the aggregated cells had low caspase-8 activity regardless of suspension conditions, whereas the single cells had higher caspase-8 activity that increased with an increasing degree of aggregation. These results suggest that cell-cell interactions in cellular aggregates of suspended MDCK cells facilitate anoikis, causing more apoptosis in individual cells than when these interactions are absent.