Proteins of the LIM family are critical regulators of development and differentiation in various cell types. Here we examined the roles of one new member of LIM family, hhLIM, in cardiac hypertrophic growth and cardiac muscle-specific gene expression. To model the increase in endogenous hhLIM transcriptional activity that occurs in response to hypertrophic stimulation, hhLIM was overexpressed using a recombinant plasmid for hhLIM. The results showed that overexpression of hhLIM resulted in increased cell volume in both C2C12 muscle cells (>1.5-fold) and cardiac myocytes (>2.49-fold), a phenotype commonly associated with cardiac hypertrophy. RT-PCR and Western blot showed that transfection of hhLIM into C2C12 muscle cells and cardiomyocytes increased skeletal alpha-actin levels and triggered the expression of the embryonic-related gene BNP, which is associated with cardiac hypertrophy. Inhibition of hhLIM expression by antisense transcripts blocked the induction of skeletal alpha-actin and BNP expression by endothelin-1. These data indicated that hhLIM played a role in regulation of cardiomyocyte growth and cell size in response to hypertrophic stimuli through its modulation of skeletal alpha-actin and BNP expression. We also determined by confocal laser scanning microscopy and immunoprecipitation that hhLIM was associated with alpha-actin and localized in the cytoplasm in unstimulated cells, and was relocalized from the cytoplasm to the nucleus upon hypertrophic stimulation. These studies suggest that hhLIM protein is involved in cardiac hypertrophy.