Fatty acids have been shown to cause death of rat and human primary pancreatic beta cells and of insulin-producing cell lines. These studies focused mainly on saturated and monounsaturated FA such as palmitic, stearic and oleic acids. In this study, we have performed a comparison of the toxicity of a wider range of FA. The toxicity of different FA to insulin-producing RINm5F cells was assessed by flow cytometry measuring loss of plasma membrane integrity and increase in DNA fragmentation. Additionally, the FA induced neutral lipid accumulation and the FA composition were determined. Palmitic, linoleic, gamma-linolenic, oleic, stearic, and eicosapentaenoic acid caused DNA fragmentation of insulin-producing RINm5F cells. Loss of membrane integrity was mainly caused by linoleic and gamma-linolenic acid. There was no correlation between cytotoxicity and the abundance of the FA in the cells as determined by HPLC analysis. Taken as whole, the toxic effect of the FA on insulin-producing RINm5F cells varied irrespective of the chain length and the degree of unsaturation. In these cells PA and LA exhibited the highest toxicity, whereas AA was not toxic. In addition, the toxicity of most tested FA was inversely related to low NLA, except for AA and EPA. The results of this study contribute to the understanding of the role of FA in the impairment of pancreatic beta cell function that occurs in type 2 diabetes and obesity.