Hepatic stellate cells (HSCs) are the major site of retinoid storage, and their activation is a key process in liver fibrogenesis. We have previously shown that expression of the retinoic acid receptor alpha (RARalpha) is upregulated in activated rat HSCs at a posttranscriptional level and that these RARalpha proteins showed a speckled distribution in the cytosol, despite their possession of a nuclear localization signal (NLS). In this report, we further characterize these cytosolic RARalpha proteins by using exogenously expressed RARalpha protein fragments or mutants tagged with a green fluorescent protein. Substitution of four amino acids, 161-164 from lysine to alanine, abolished the NLS. Exogenously expressed RARalpha protein fragments containing an NLS were localized exclusively in the nuclei of activated rat HSCs and never colocalized with the endogenous RARalpha proteins in the cytosol, suggesting that the NLS of endogenous RARalpha proteins is masked. Biochemical analysis showed that 65% of RARalpha proteins in activated HSCs were insoluble in a mixture of detergents. The insolubility of RARalpha proteins makes it difficult to identify RARalpha proteins in activated HSCs. Therefore, we propose that insoluble, speckled cytosolic distribution of RARalpha proteins represents a new marker of HSC activation.