For coniferous gymnosperms, few data exist as to the contribution of the membrane-associated proteome to cell wall and wood formation. In this study, we begin to address this knowledge deficiency by examining the proteomic profile of Golgi-enriched membrane preparations derived from developing Pinus radiata compression wood. These membrane preparations were generated by a combination of discontinuous sucrose gradient centrifugation and Triton X-114-based phase separation. Fractionation by phase separation removed contaminating proteins associated with the cytoskeleton and enabled the discrimination between soluble and membrane-bound/integral proteins. The proteomic analysis of the resulting aqueous and detergent phases using high-performance liquid chromatography-tandem mass spectrometry resulted in the identification of 175 proteins. The majority of the identified proteins were membrane bound/integral and originated from cellular components such as the nucleus, plastids, endoplasmic reticulum, plasma membrane and Golgi vesicles. On the basis of bioinformatic analysis, many of the identified proteins were predicted to be involved either in the regulation of wood formation or in cell wall biosynthesis, which indicated that the proteomic analysis of non-cytosolic proteins in developing xylem is a useful strategy to investigate the molecular aspects of wood formation in pine.