A very convenient method to quantify coliform bacteria in water can probably be designed via the determination of the activity of the enzyme beta-D-galactosidase, whose natural occurrence is, apart from less frequently occurring aeromonads mainly restricted to this type of microorganisms. 4-methylumbelliferyl-beta-D-galactoside is used as substrate, which is hydrolyzed during the enzymatic reaction; the released 4-methylumbelliferone can be quantified fluorimetrically. In the present study the influence of various physical and chemical parameters on the determination is investigated and the experimental conditions are optimized. Most important entities are the pH value during hydrolysis, the presence of nutrients and co-factors in the sample, and the modification of the substrate. Statistical evaluation of the results obtained by changing single or multiple parameters reflects clearly their positive or negative influence on the enzyme activity. Thus, deliberate addition of surfactants, specific nutrients, salts and co-enzymes results in a significantly increased activity of beta-D-galactosidase towards the substrate, which can be advantageously exploited to increase the sensitivity of the analytical method together with a decrease of the detection limit. The influence of the parameters and the optimized conditions of the improved analytical methods are presented.