We have previously shown that a dialyzed, lyophilized saline extract from bovine urothelium can restore the antiadherence activity of the rabbit bladder following mucin removal with 50% acetone. This report describes results of initial purification of antiadherence factor(s) from bovine bladder mucin and describes results of biochemical analysis in an attempt to elucidate possible mechanism of this antiadherence activity. Separation performed by gel filtration (Spectra gel AcA 34, 20-350 KD range) results in three fractions. Only the low molecular weight fraction had a statistically significant inhibitory effect on bacterial adherence to the mucin deficient rabbit bladder. After overnight dialysis against running deionized water and lyophilization, the crude extract contained 60% protein while gel filtration fractions 1-3 contained 35%, 90% and 15% by weight respectively. The first fraction (apparent high molecular weight, greater than 350 kD) did not appear to enter SDS polyacrylamide electrophoresis gels (SDS-PAGE, 3-12%) or agarose gels (0.5%) to any significant extent. In the fractions that displayed antiadherence activity (the crude extract, fractions 2 and 3) SDS-PAGE bands were seen corresponding to an apparent molecular weight of 78 kD in addition to bands co-migrating with bovine serum albumin (BSA). BSA itself slightly increases bacterial adherence in this model. Most of the albumin of the crude extract was found in the second fraction (60%). On the other hand most of the sulfate and sugar of the crude extract was found in the third, low molecular weight fraction. Since sulfated polysaccharides such as heparin and dextran sulfate are very effective antiadherence agents in this rabbit bladder model, it is conceivable that the sulfated sugar content of the third fraction is responsible for its antiadherence effect on the mucin deficient rabbit bladder.