Hsp20, a recently described new member of the small heat shock protein superfamily, is abundant in heart, skeletal muscle types and smooth muscle. We investigated the intracellular localization of Hsp20 in cultured rat neonatal cardiac myocytes, under normal conditions and after stress. These cellular characteristics of Hsp20 were compared with those of its closest relative, alphaB-crystallin, which is also highly expressed in heart. Like alphaB-crystallin, Hsp20 is normally located in the cytoplasm of the cardiac myocytes. After a heat stress, a subpopulation of Hsp20 migrates into the nucleus, while another part remains in the cytoplasm. In very few cells a faint sarcomeric association of Hsp20 is observed. In contrast, as previously reported, alphaB-crystallin displays a very distinct cross-striated sarcomeric staining after the heat shock, but no nuclear migration. Also at the level of Triton solubility, differences exist between the two related proteins; while alphaB-crystallin, like other small heat shock proteins, becomes insoluble upon heat stress, Hsp20 remains largely soluble. Our results indicate that Hsp20 and alphaB-crystallin, despite their structural similarities, display conspicuous functional differences.