Molecular methods for HIV-1 infection using dried blood-spot (DBS) for HIV-1 CRF01_AE subtypes have not been fully optimized. In this study assays for HIV-1 diagnosis or quantitation were evaluated using infant DBS from Thailand. Paired DBS and whole blood samples from 56 HIV-1 CRF01_AE or B'-infected infants were tested for infant diagnosis using modified Amplicor DNA PCR and NucliSens RNA NASBA and an in-house real-time PCR assay. The Amplicor Monitor viral load (VL) assay, with modifications for DBS, was also evaluated. DBS VL were hematocrit corrected. Stability studies were done on DBS stored at -70 degrees C to 37 degrees C for up to 1 year. The DBS diagnostic assays were 96-100% sensitive and 100% specific for HIV-1 diagnosis. DBS HIV-1 VL were highly correlated with plasma VL when corrected using the actual or an assumed hematocrit factor (r(c)=0.88 or 0.93, respectively). HIV-1 DNA in DBS appeared to be more stable than RNA and could be detected after up to 9 months at most temperatures. DBS VL could be consistently determined when stored frozen. These results show that DBS can be used accurately instead of whole blood for the diagnosis of HIV-1 infection and VL quantitation, particularly if samples are appropriately stored.