The NanoChip system was used for subtyping human immunodeficiency virus type 1 (HIV-1) strains using probes complementary to the V1 region of the env gene. Probes for six subtypes (A to D, F, and G) and two circulating recombinant forms (AG and AE) of HIV-1 group M were included. The specificity of these oligonucleotides had been evaluated previously in a DNA enzyme immunoassay. Samples from 112 patient sera were used as templates in a nested reverse transcription-PCR to produce amplicons that were applied to the array. The array was then hybridized successively to pairs of oligonucleotide probes. The strains were assigned a subtype on the basis of their probe hybridization patterns. One strain gave a contradictory pattern and was designated as untypeable by the NanoChip assay. Eighty-eight strains gave hybridization patterns that allowed a correct subtype designation to be made by the NanoChip assay compared to either the sequence or the heteroduplex mobility assay (HMA)-determined subtypes. Thirteen strains that reacted with the subtype A probe (SA2) were incorrectly assigned to subtype A, or to one of the related circulating recombinant types (AE or AG), on the basis of reactions with probe SAE1 or SAG1. The results indicate that these oligonucleotides have relatively low specificities. The probe subtypes of three strains matched the subtypes determined for the gag and pol genes but not the env gene, suggesting that a recombination event may have occurred within the env gene. Overall, the NanoChip assay gave results comparable to those for HMA and sequencing and provides a convenient and cost-effective means by which to subtype HIV-1.