Spontaneous variation of the level of alginate synthesis in Pseudomonas aeruginosa was associated with changes in the activity of all four enzymes leading to synthesis of GDP-mannuronic acid, the activated precursor for polymerization. For the high-alginate-producing variant 8821M, alginate yield and properties, as well as the levels of alginate enzymes, were dependent on growth temperature. In contrast, levels of alginate and enzymes in the mucoid parent strain 8821 were very low and near temperature-independent. The difference in the specific activity of GDP-mannose dehydrogenase (GMD), encoded by the algD gene, between the two strains was associated with the alginate biosynthetic ability and with the degree of activation of the algD promoter, measured using the algD-xylE transcription fusion on plasmid pVD2X. Maximal activity of the four enzymes was observed in strain 8821M grown at 30 degrees C, a temperature below the optimum for growth (35 degrees C). The effect of temperature on GMD activity could not be explained by the regulation of the algD promoter by temperature, since expression of pVDZX appeared to be more active at 35 degrees C, when the decrease of pVD2X copy number with increasing temperature was taken into account. The involvement of enzymes that catalyse steps downstream from the formation of the activated precursor should also be considered, as suggested by differences in the molecular mass of alginates synthesized by the two strains at various temperatures. Acetyl content of alginates increased as temperature decreased and strain 8821M produced the highest levels of acetylated polymers. The degree of acetylation appeared to be related to growth rate and could reflect acetyl-CoA availability.