The toxic effects of silver nanoparticles (AgNPs) on cells are well established, but only limited studies on the effect of AgNPs and silver ions on the cellular transcriptome have been performed. In this study, the effect of AgNPs on the gene expression in the human lung epithelial cell line A549, exposed to 12.1 µg/ml AgNPs (EC20) for 24 and 48 h was compared with the response to control and silver ion (Ag+) treated cells (1.3 µg/ml) using microarray analysis. Twenty-four hours exposure to AgNP altered the regulation of more than 1000 genes (more than 2-fold regulation); whereas considerably fewer genes responded to Ag+ (133 genes). The up-regulated genes included members of the metallothionein, heat shock protein and histone families. As expected from the induction of metallothionein and heat shock protein genes, Ag+ and AgNP treatment resulted in intracellular production of ROS, but did not induce apoptosis or necrosis at the concentrations used in this study. In addition, the exposure to AgNPs influenced the cell cycle and led to an arrest in the G2/M phase as shown by cell cycle studies by flow cytometry and microscopy. In conclusion, although the transcriptional response to Ag+ exposure was highly related to the response caused by AgNPs, our findings suggest that AgNPs, due to their particulate form, affect exposed cells in a more complex way.