To understand the importance of substratum in terms of biofilm formation, this study studied differences in the proteins produced by Pseudomonas putida strain F1 grown under different conditions. Specifically, this asked what proteins were made when biofilms of P. putida were formed on steel mesh balls and glass wool. Two-dimensional gel electrophoresis analysis of total soluble proteins allowed both sets of biofilm-grown cells to be compared with those of planktonically-grown cells. After analysis by Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry, fifteen differentially expressed proteins were identified and grouped into several classes: proteins involved in metabolism, transportation, adaptation or protection, biosynthesis and other functions. Five proteins showed differential expression only on one substratum. Enolase, a protein with multiple functions in prokaryotic cells, was up-regulated in mature biofilms both at protein level and mRNA level, indicating the protein expression was regulated in transcription level. These findings will help to understand the development of biofilms and the role of the substratum in this process.