In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.