The effects of ethanol/broth proportions and the number of steps at varying pH in the presence or absence of sodium chloride (NaCl) were studied as precipitation strategies for the recovery and purification of high molar mass bio-hyaluronic acid (Bio-HA). Bio-HA was synthesized by Streptococcus zooepidemicus in a culture medium containing glucose and soy peptones. A single-step precipitation was more attractive than multistep precipitation in terms of recovery and purity as well as decreased use of ethanol. The best conditions in the absence and presence of salt were 2:1 ethanol/broth (v/v) at pH4 (55.0 +/- 0.2% purity and 85.0 +/- 0.7% recovery) and 2:1 ethanol/broth (v/v) at pH7+2molL(-1) NaCl (59.0 +/- 0.9% purity and 82.0 +/- 4.3% recovery). Dynamic light scattering (DLS) spectra showed different particle sizes as a consequence of the changes in the molecular structure of HA, mainly with changes in pH. Although slight changes in distribution were observed, the average HA molar mass was not affected by the precipitation strategy, remaining on the order of 10(5)Da. Therefore, pH and NaCl modulated the precipitation performance of HA. These findings are relevant to further optimizing the precipitation step, thus minimizing costs in the later stages of HA purification.