An exopolysaccharide depolymerase was isolated from a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin. Purified preparations of the depolymerase showed maximum activity against the unacetylated polymannuronic acid exopolysaccharide from the same strain and little activity against commercially prepared alginic acid. The evidence suggests that the enzyme is either periplasmic in location or associated with the outer cell membrane and is released extracellularly, in the absence of cell lysis, after a reduction of the culture magnesium (Mg2+) concentration below 3.0 mM. The depolymerase is also released after the addition of sublethal concentrations of EDTA to cultures containing 3.0 mM Mg2+. A survey of additional mucoid P. aeruginosa isolates recovered from patients with cystic fibrosis showed that nearly 60% demonstrated similar depolymerase activity while none of the nonmucoid revertants of the parent strains produced detectable depolymerase activity.