Biliary and gallstone sterol compositions were analyzed in 20 consecutive cholecystectomized patients. The main intention was to identify and quantitate noncholesterol sterols and to compare the sterol patterns of the two sources. Cholesterol comprised approximately 97% of the stone and gallbladder bile sterols; the remainder were from plant sterols, cholestanol, and cholesterol precursors, mainly lathosterol and methylsterols (two methostenols, lanosterol, and two dimethylsterols). Desmosterol and delta 8-lathosterol were also identified in both the bile and the gallstones. The sterol patterns of the bile and gallstones differed markedly. Thus, the contents of the two lathosterols and the two methostenols were clearly higher in the gallstones, whereas lanosterol stayed almost totally, and other minor sterols were preferentially, in the bile. In fact, the gallstone methylsterols consisted mainly of the two methostenols, a pattern usually seen in esterified methylsterols in serum. The core and matrix of the stone, and large and small stones as well, had only a small variation in their sterol composition within each individual, suggesting that the pattern of the noncholesterol sterol precipitation remains the same during the growth of the stone. Centrifugation of the bile revealed sedimentation of methylsterols with the stonelike sterol pattern. It can be speculated that the soluble and poorly soluble bile sterols have different hepatic origins and that the similarity between (a) methylsterols in the stone and sediment and (b) esterified methylsterols in serum points to a common hepatic site of origin.