Nucleation time represents the terminal step in in vitro studies examining bile lithogenicity. Because of the concern that residual microcrystals, left after ultracentrifugation, may be responsible for the rapid nucleation time of gallbladder bile from patients with cholesterol gallstones, we have included a final filtration step. However, we found this procedure to considerably lengthen the nucleation time of abnormal biles. In view of the central importance of the nucleation assay we compared the effect of three commonly used gallbladder bile pre-treatment regimes (designed to remove endogenous crystals) on nucleation time. They were: a) immediate filtration of bile (0.22 micron filter); b) ultracentrifugation; and c) ultracentrifugation followed by filtration. The respective nucleation times were: a) 9.3 +/- 3.7 days, n = 6; b) 2.9 +/- 0.4 days, n = 10; c) 12.8 +/- 2.3 days, n = 11. To determine whether the dramatic change in nucleation time was due to the removal of components other than seed crystals, we examined the mucus content, the total lipid composition of bile, and that of its cholesterol transport components following the different pre-treatments. No significant difference in total lipid, percentage cholesterol carried by the transport components, or their cholesterol/phospholipid ratio were found. Ultracentrifugation alone was sufficient to removal all detectable large molecular weight mucus glycoprotein. Although nucleation time of the abnormal gallbladder samples was extended in the ultracentrifuged/filtered biles, it was still significantly different (P less than 0.01) from that of normal gallbladder biles, confirming an intrinsic difference between abnormal and normal biles, in cholesterol metastability. We also examined the effect of protein digestion on the nucleation time of native biles.(ABSTRACT TRUNCATED AT 250 WORDS)