Pseudomonas fluorescens strain CL0145A was discovered at the New York State Museum Field Research Laboratory as an effective agent against the environmentally destructive zebra mussel, which has contaminated US waters. Dried cells of the microbe are being commercialized as an environmentally friendly solution to the problem. We found that antibiotic activity against the Gram-positive bacterium Bacillus subtilis is produced and excreted by this strain. We have carried out studies to optimize production of the antibiotic. Studies were begun in a complex corn meal medium. Activity was found in both cells and culture supernates and was maximal after one day of fermentation. Static fermentation conditions were found to be superior to shaken culture. Production of extracellular antibiotic in complex medium was found to be dependent on the content of sucrose and enzyme-hydrolyzed casein. Indeed, production was greater in sucrose plus enzyme-hydrolyzed casein than in the complex medium. Of a large number of carbon sources studied as improvements over sucrose, the best was glycerol. An examination of nitrogen sources showed that production was improved by replacement of enzyme-hydrolyzed casein with soy hydrolysates. Production in the simple glycerol-Hy-Soy medium was not improved by addition of an inorganic salt mixture or by complex nitrogen sources, with the exception of malt extract. In an attempt to keep the medium more defined, we studied the effect of amino acids and vitamins as replacements for malt extract. Of 21 amino acids and 7 vitamins, we found tryptophan, glutamine, biotin, and riboflavin to be stimulatory. The final medium contained glycerol, Hy-Soy, tryptophan, glutamine, biotin, and riboflavin.