Hepatitis A virus (HAV) released from infected BS-C-1 cells was incompletely neutralized when incubated with a variety of convalescent sera (non-neutralizable fraction of 17 to 32%). Chloroform extraction of virus resulted in a substantial reduction of the non-neutralizable fraction (to less than 1%), suggesting that non-neutralizable virions might be associated with lipids. Non-neutralizable HAV recovered from untreated cell culture supernatant fluids sedimented heterogeneously and less rapidly than normal virus in rate-zonal sucrose gradients and also banded at a lower density in CsCl (1.14 to 1.18 g/ml) than normal, neutralizable virus (1.32 g/ml). This bimodal distribution of HAV in CsCl gradients was confirmed by cDNA-RNA hybridization. Together, these observations suggest that a substantial proportion of HAV particles released from infected cells are lipid-associated and imply an important role for cell membranes in the assembly and release of HAV in vitro.