Because beef contains a high proportion of marbling, we sought to optimize the solubilization of the longissimus muscle proteome using modified lysis and isoelectric focusing (IEF) rehydration buffers. Of the 3 lysis buffers tested, those containing a thiourea-urea mixture provided superior resolution, whereas that which contained only urea yielded consistently poor results. In addition, we found that the IEF rehydration buffer containing a thiourea-urea mixture, a high dithiothreitol level, and Pharmalyte was able to generate sharper 2-dimensional (2-D) gels, whereas that which contained immobilized pH gradient (IPG) buffer had no an effect on protein resolution. The number of different protein spots between the muscle-development stage (11 months) and the fat-development stage (17 months) in Hanwoo steers was 39, whereas the use of normal buffer resulted in a detection of a difference of only 7 protein spots. This study provides a methodological tool for studying the proteome of bovine longissimus muscle with a high fat content using 2-D gel electrophoresis (2-DE) gels.