Current methods of forensic DNA profiling by restriction fragment length polymorphism analysis rely on radioactive detection of DNA. The use of radioactive isotopes is complicated, expensive and requires elaborate safety precautions. Recently, non-radioactive detection methods involving the use of alkaline phosphatase-conjugated oligonucleotide probes have become available. These probes differ from most 32P-labeled probes in that they are synthetic oligonucleotides, whereas the 32P-labeled probes are purified plasmid inserts from cloned VNTR regions. Because of this difference, it is possible that the specificity of the non-isotopic probe will differ from that of the 32P-labeled probe. This study compares the specificity of the two types of probes by parallel analysis of a set of DNA samples, including a subset of relatively small alleles, at the loci D1S7, D2S44, D4S139, D10S28, and D17S26. We found that although the alkaline phosphatase detection method is slightly less sensitive than 32P detection, the AP-conjugated oligonucleotide probes tested have specificity comparable to, and are appropriate and suitable substitutes for, 32P-labeled plasmid inserts.