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One of the most useful tools in cell biology research is introduction of foreign materials (such as nucleic acid) into eukaryotic cells. Transfection of mammalian cell lines with DNA is one of the most popular techniques to achieve this. The traditional strategy for achieving this is via a calcium phosphate based co-precipitation with nucleic acid. Further, in much of today's research there is a growing need to transfect large quantities of cells. The use of calcium phosphate is ideally suited for this due to its relatively good efficiency, scalability and, most importantly, its low cost. The utility of the Corning High Yielding Performance Flask (HYPERFlask) cell culture vessel includes its performance in routine transfection procedures. To this end we have developed a robust protocol that can be applied and modified if necessary for your needs. The protocol was developed using Chinese hamster ovary (CHO) cells but has been successfully applied to a variety of other cell types including HeLa cells. The protocol is intended as a starting point to optimize your work. We used a Calcium Phosphate Transfection Kit (Invitrogen Cat. No. K2780-01) in developing this protocol.