We have compared the protein maps of agar-entrapped Pseudomonas aeruginosa cells to those of free counterparts grown in the presence or absence of the immobilized-cell gel support. Principal component analyses (PCAs) were used to interpret spot quantity variations observed on electropherograms obtained by two-dimensional gel electrophoresis. PCA of the data matrix (923 rows x 6 columns) in which spot density values were standardized horizontally extracted three principal components (PCs) with eigenvalues higher than 1, accounting together for 71.6% of the variability in the data. Principal component 1 (PC1) opposed free (F) and agar-entrapped (AE) cultures, with a low contribution of agar-released, free (ARF) cultures to PC1. Inversely, the contribution of ARF cultures to PC2 was high, opposing those of AE and F cultures. Component 3 was related to the duration of incubation. Only 10% of total proteins were upregulated in AE cells during the first 18 h of incubation, the number of underexpressed peptides balancing that of overexpressed ones. Downregulation clearly became the dominant tendency when the incubation time was extended to 48 h. These results demonstrate that AE and ARF bacteria are physiologically different from F organisms.