Alkaline phosphatase (APase) activity of Megasphaera elsdenii was enhanced by PO42− limitation in batch culture; however, six other species of rumen bacteria tested showed no increase in APase activity under these conditions. Alkaline phosphatase was produced by the mixed rumen microflora even though the inorganic phosphorus concentration was as high as 10 mM. The APase activity of the bacterial fraction from rumen fluid was not increased during incubation in a phosphorus-free culture medium. Since bacteria may account for greater than 80% of the APase activity in the rumen, this would suggest that the bulk of the APase activity in the rumen is synthesized constitutively. The bacterium responsible for most of the APase activity probably is Bacteroides ruminicola.