In this study rat dermal fibroblasts (RDFs) were cultured on smooth or microgrooved (1-20 m wide, 0.5-5.4 m deep) substrates. Polystyrene microgrooved substrates were produced by solvent casting on molds that had been produced by photolithographic techniques. We investigated the attachment of RDFs with various analytical techniques. Light microscopy and image analysis showed that RDFs were oriented on most microgrooves. The rate of orientation effectively was increased by an increase of groove depth. An analysis of confluent layers of RDF showed that at confluency microgrooves were able to support greater numbers of cells. However, the largest numbers of cells were not found on the narrowest and deepest microgrooves even though such microgrooves have the largest total surface and induce the strongest alignment. Interference reflection microscopy (IRM) showed that the RDFs form focal adhesions where the cell membrane is only 10 nm from the substrate. IRM also showed that RDFs follow the contours of shallow and wide microgrooves but bridge the grooves on deeper and narrower ones. This could explain why such grooves are not able to increase the numerical cell adhesion to a greater degree. The absence of contact between cells and the bottom of the grooves is a very important factor in establishing contact guidance.