This study utilizes a surface plasmon resonance (SPR) biosensing and a theoretical secondary structure calculation to investigate the influence of secondary structures on the DNA hybridization. It is found that the SPR angular shifts associated with the three pairs of 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two pairs of oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35 to 45 degreesC reduces secondary structure effects. On the hybridization with mixture target oligonucleotides, the SPR results demonstrate that secondary structures interfere significantly. (C) 2004 Elsevier B.V. All rights reserved.