We have attempted to develop surface-enhanced Raman scattering (SERS) substrates by the use of two-dimensional (2D) Au nanorod arrays and to characterize the SERS-active sites of the Au nanostructures. We prepared two different types of 2D Au nanorod arrays by means of the anodic aluminum oxide (AAO) template-assisted nanofabrication. In the template-embedded array (type I) an Au nanorod fills up the AAO pore, while in the annularly spaced array (type II) an Au nanorod keeps approximately 20 nm away from the pore wall. The strongest SERS effect was observed for both types of substrates with an Au nanorod diameter of approximately 66 nm. With respect to the bare AAO template, the NaOH-etched (unetched) type I substrate with the 66 nm Au nanorod showed 115-fold (63-fold) enhancement in Raman scattering, corresponding to a SERS enhancement factor of 10(7)-10(9). For the unetched and ordered Au naonorod arrays, the SERS-active sites localized around the rod end, whereas for the NaOH-etched and partially aggregated Au nanorods, hot spots for SERS were found in the interstices between rod tips. The type II substrate allowed the effect of rod length on the SERS response to be investigated, and the SERS response was observed to vary very little with the rod length increase (250-1000 nm), indicating that majority of the signal originates at the rod end. A comparison between the analytes all-trans-beta-carotene and pyridine suggests that, for both types of substrates, the electromagnetic enhancement is predominant over the chemical enhancement. This work demonstrates that the unetched type I substrate is highly SERS effective, for which the fabrication protocol is advantageous in its simplicity and reproducibility.