Identification of genes differentially expressed during water-deficit stress is an important step for developing strategies to improve drought tolerance of cultivated upland cotton (Gossypium hirsutum L.). The objective of this study was to develop a model of the responses that lead to drought tolerance in roots of the drought tolerant cultivar, Siokra L23. A polyethylene glycol (PEG)-induced phenotype derived from leaf measurements revealed a decline in the photosynthetic rate and an increase in leaf temperature between 0 and 48 h, followed by a return to initial values from 48 to 96 h after stress imposition. Due to the biphasic nature, these two phases were designated as response and recovery, respectively. Root sugar content decreased 40% at 24 h, indicating carbon limitation. Organic acid levels, however, increased or remained unchanged suggesting a shift to more efficient pathways for production of carbon skeletons. Osmotic adjustment was a component of the adaptive response indicated by a 29% increase in K+ content in stressed roots at 24 h. Profiles of root gene expression were developed at 0, 1, 4, 24, and 96 h after stress initiation utilizing cotton fiber oligonucleotide microarrays to gain insight into the cellular processes that lead to recovery. Genes from the glyoxylate cycle and two genes involved in K+ transport were identified as candidate genes involved in root-cell osmotic adjustment. The solute accumulation was coupled to simultaneous reductions in the expression levels of seven aquaporins. Regulation of membrane permeability to water, decreased cellular solute potential, and efficient production of carbon skeletons are probably the primary root-localized mechanisms by which Siokra L23 adapts to PEG-induced osmotic shock.