In the past, research on the development of cotton fiber was hampered by problems in separating cotton fiber proteins by electrophoresis. An improved method of sample preparation for two-dimensional electrophoresis of proteins from cotton fiber cells was developed. The phenol extraction method of Hurkman and Tanaka (Plant Physiol. 81:802-806) was modified to extract the total protein from fiber cells. Samples were ultracentrifuged before loading onto electrophoresis gels. This procedure overcomes problems encountered in the past by eliminating secondary cellular metabolites that interfere with the electrophoretic separation of proteins. This method was used to compare the proteins in cells synthesizing primary cell walls (14 days post-anthesis, DPA) and cells synthesizing secondary cell walls (21 DPA). The method of Hochstrasser et. al. (Anal. Biochem. 173: 424-435) was used for the two-dimensional electrophoresis. Proteins which consistently showed large quantitative differences between 14 and 21 DPA were identified. Eight proteins were identified that were present in much higher quantities in the 21 DPA fiber than in 14 DPA fiber. Sixteen proteins were identified that were present in much higher quantities in the 14 DPA fiber than in the 21 DPA fiber. Further analysis of these proteins will provide new insight into fiber development.