Biofilms are aggregates of cells adhering to surfaces embedded in a matrix of extracellular polymeric substances of their own making. Microbial water availability in many terrestrial habitats is one of the most important factors influencing unsaturated biofilm development and biofilm cell survival and death. In general, Pseudomonas putida strain mt-2 unsaturated biofilm formation proceeds through three distinct developmental phases, culminating in the formation of a microcolony. The form and severity of reduced water availability alters cell morphology. The dehydration treatments resulted in biofilms comprised of smaller cells but they were taller and more porous, and had a thicker exopolysaccharide (EPS) layer at the air interface. In the osmotic stress treatments, cell filamentation occurred more frequently in the presence of high concentrations of ionic, but not non-ionic, solutes and these filamented cells drastically altered biofilm Architecture;P. putida produced more EPS in response to dehydration stress, but not thermodynamically equivalent osmotic stress. Carbohydrate composition analysis showed that alginate was a component of unsaturated biofilm EPS only in the presence of dehydration stress. The absence of alginate altered biofilm architecture in that biofilms were shorter, covered more surface area, and were less porous than when alginate was present. By measuring intracellular water potential changes and monitoring fatty acid alterations during dehydration stress, we demonstrated that alginate slows the rate of cellular dehydration. Alginate production also contributed to the ability of cells to survive a severe desiccation stress. These results suggest that an important consequence of cellular dehydration is alginate production, which contributes to the fitness of P. putida in water-limited environments.;Reduced water availability influenced the temporal and spatial localization of dead cells within unsaturated biofilms. Dead cells were organized in arrays of various lengths one cell width. In general, more dead cells were localized in the lower layers, while active, growing cells were localized primarily in the upper layers.;Taken together, reduction of water availability influences biofilm architecture, EPS production, and cell death patterns, which all reflect a status of adaptation of P. putida to dehydration stress. This study provides new insights into microbial dehydration physiology.