The binding of human DNA polymerase beta (pol beta) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol beta inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol beta inhibitors. In the interaction between pol beta and DNA, pol beta could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol beta inhibitors prevented the binding of pol beta to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol beta to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol beta-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol beta to DNA was mainly due to 7-fold increase in the dissociation rate.