Efficient preparation of DNA templates is an important step in large-scale DNA sequencing. The ensure high-quality sequence data, we have prepared M13 phage DNA templates using a glass fiber-filtration method. We present the adaptation of this protocol to a 96-well format using commercially available filter plates. Two variations are described: one using polyethylene glycol precipitation and a second where the phage particles are disrupted before filtration, thus eliminating the need for precipitation. Using either of these protocols, 96 templates can be prepared in less than 2 h. Sufficient DNA for 1-2 dye primer sequencing reactions is routinely obtained from 1 mL of culture, and the resulting sequence data are of high quality.