Introduction: cDNA microarray technology has greatly facilitated mechanistic studies in pharmacology and toxicology. A clean hybridization with minimal background is critical for successful microarray analysis and is highly desired. However, clean hybridization alone is not enough; verification is needed. Methods: Total RNA was isolated from the livers of acetaminophen-intoxicated mice and was subjected to cDNA microarray analyses using ExpressHyb, ULTRArrayHyb or MicroHyb on nylon membranes. Real-time RT-PCR analyses were performed for verification. Results: We have demonstrated in this paper that hybridization systems can significantly impact the quality of array data. MicroHyb produced very clean hybridizations, but some results could not be confirmed by real-time RT-PCR and in accord with biological responses. The hybridization images from ExpressHyb were not always clean, but were reliable. The sensitivity of ULTRArrayHyb was moderate. Conclusion: This study has indicated the importance of selecting hybridization buffers in membrane arrays and recommended real-time RT-PCR for follow-up analysis. Gene expression changes should also be correlated with biological significance.