Dietary fats have been implicated in the progression of coronary heart disease and clear differences have been shown for the effects of saturated, monounsaturated and polyunsaturated fatty acids on the different stages of disease. In part, these effects can be explained by the regulation of gene transcription. In this study, a novel cardiac cell-line was targeted for the investigation of fatty acid effects on gene transcription. The aims of this study were to (i) characterise the P19CL6 cell-line at the transcriptional level, (ii) investigate the effects of long-chain fatty acids and clofibrate on mRNA levels of specific lipid metabolism-related genes, such as heart-type fatty acid-binding protein and peroxisome proliferator-activated receptor-α, -β, and -γ in the P19CL6 cell-line, and (iii) determine the effects of long chain fatty acids and clofibrate on global transcriptome levels in P19CL6 cells, using cDNA microarray analysis. P19CL6 cells displayed characteristics indicative of a cardiomyocyte phenotype including: the expression of cardiac-specific markers (α-MHC, β-MHC and CaCh-h), an increase in pulse rate with increasing adrenaline concentration and the display of mononuclear cardiomyocyte morphology. However, spontaneous contraction was inconsistent between cultures of P19CL6 cells and pulse rate decreased significantly with increasing passage number until no “beating” was eventually recorded. These observations led us to conclude that P19CL6 cells may display a heterogeneous phenotype, which was investigated further. Analysis of microarray data indicated that global transcriptome profiles observed in the P19CL6 cell-line are not indicative of a pure adult cardiac or skeletal muscle phenotype, and more closely related to mouse embryonic heart tissue. However, the H9C2(2-1) cell-line was more closely related to adult and embryonic heart tissue than the P19CL6 cell-line. Therefore it was concluded that the H9C2(2-1) cell-line represents a better 'cardiomyocyte model system than the P19CL6 cell-line. The results of this study also showed that in P19CL6 cells long-chain fatty acids (but not clofibrate) significantly increased the abundance of peroxisome proliferator-activated receptor-α and -γ, whereas no changes were observed in the expression levels of heart-type fatty acid-binding protein and peroxisome proliferator-activated receptor-β, under the conditions used. The non-response of the latter genes can probably be explained by the relatively short time of exposure of cells in culture. Microarray analysis showed that linoleic and α-linolenic acids and clofibrate had similar effects, and that these differed from those of palmitic and oleic acids. These results are in agreement with other studies showing that cellular responses to polyunsaturated fatty acids differ from those observed with saturated and monounsaturated fatty acids.