Hyaluronic acid (HA), a linear polysaccharide composed of alternating glucuronic acid and N-acetylglucosamine residues, is an essential molecule of higher vertebrates. The fowl cholera pathogen Pasteurella multocida Carter Type A also produces HA in the form of an extracellular capsule in order to evade host defenses. HA synthase activity could be obtained from cell-free membrane preparations of P. multocida. The enzyme utilized UDP-sugar precursors of HA in the presence of Mg2+ or Mn2+ at neutral pH. Mn2+ at 1 mM stimulated approximately 2-fold more incorporation than Mg2+ at 10 mM. On the other hand, the analogous enzyme from group A Streptococcus, HasA, is stimulated more by Mg2+ than Mn2+. The apparent Michaelis constants, K(M), of the P. multocida HA synthase for UDP-N-acetylglucosamine and UDP-glucuronic acid were estimated to be approximately 75 and approximately 20 microM, respectively, in the presence of Mg2+, which suggests that the substrates are bound with 2-3-fold higher affinity than by the HasA enzyme. The rate enhancement observed with Mn2+ is apparently not due to better binding of the sugar nucleotide precursors complexed to Mn ion because the K(M) value, a measure of substrate affinity, increases by 25-50% in comparison to Mg2+. In summary, the HA synthase from P. multocida, a Gram-negative bacterium, has kinetic optima distinct from those of HasA, the analog from the Gram-positive group A Streptococcus.