Our aim was to determine whether fractionation of human urine affects the attachment of calcium oxalate monohydrate (COM) crystals to renal cells. Urines collected from 6 healthy subjects were fractionated into sieved (S), centrifuged (C), centrifuged and filtered (CF), or ultrafiltered (UF). Attachment of ¹⁴C-COM crystals to Madin-Darby canine kidney (MDCK) cells was studied after pre-coating the crystals or the cells with the urine fractions, and by using the same fractions as the binding medium. Proteins content of the fractions and pre-coated crystals were analysed with SDS-PAGE and Western blotting. All urine fractions inhibited crystal attachment. When fractions from the 6 urine samples were used to pre-coat the cells, the median inhibitions of crystal adhesion (~40%) were not significantly different. Median inhibition after pre-incubation of crystals was the same for the S, C and CF fractions (~40%), but significantly greater than for the UF fraction (~28%). When fractions were used as the binding medium, median inhibitions decreased from 64% in the S fraction to 47% (C), 42% (CF), to 29% (UF). SDS-PAGE analysis showed that centrifugation and filtration reduced the amount of Tamm-Horsfall glycoprotein (THG), which was confirmed by Western blotting. Human serum albumin, urinary prothrombin fragment 1 and osteopontin, but not THG, were present in demineralized extracts of the pre-coated crystals. Fractionation of human urine affects the attachment of COM crystals to MDCK cells. Hence future studies investigating regulation of crystal-cell interactions should be carried out in untreated urine as the binding medium.