Low-density lipoprotein (LDL) receptor binding is the initial step in receptor-mediated clearance. Dietary fat composition is known to affect LDL clearance, but the mechanism of the effect is unknown. We have examined the effects of altered membrane fatty acid composition, as might occur when specific dietary fats are consumed, on LDL binding using a Chinese hamster ovary (CHO) line that constitutively expresses the human LDL receptor. Binding of pooled human LDL to its receptor was compared in cells enriched with various fatty acids. Binding affinity was greater (lower Kd) for cells grown in 16:0-, 18:0-, or 18:1-enriched media than for those grown in 18:2 (P < .0001). The apparent receptor number (Bmax) was lower for cells enriched in saturated fatty acids and 18:1. Fluidity was assessed by measuring diphenylhexatriene (DPH) fluorescence anisotropy (rs). Cells enriched in 18:1 or 18:2 were the most fluid (P < .003). The correlation between binding and fluidity (r = .24, P = .27) was weak and did not appear to explain the effects of fatty acid modification on LDL receptor binding. Thus, it appears that cellular enrichment in 16:0, 18:0, and 18:1 increases binding affinity by affecting properties other than membrane fluidity. Changes in Bmax may also contribute to the observed differences in LDL binding.