Plasma specimens collected in 1999 from 32 human immunodeficiency virus type 1 (HIV-1)-infected foreigners living in Madrid, Spain, were examined for the presence of non-B subtypes. Furthermore, plasma viremia was quantified using two different AMPLICOR HIV-1 MONITOR tests, version 1.0 and the new upgraded and automated version 1.5 (COBAS). Most patients came from Africa, where they most likely had acquired HIV-1 infection through sexual contact. HIV-1 genetic subtyping was based on the phylogenetic analysis of the protease gene. Twenty-two subtype B, six subtype G, two subtype C, one subtype A, and one D subtype infection were found. Overall, non-B subtypes represented 31.25% of the study population. Irrespective of the HIV-1 variant, viral load values above the detection limit (200 HIV RNA copies/ml) increased from 56.2 to 71.9% for results obtained using MONITOR version 1.0 and COBAS, respectively. Moreover, significant differences in viral load values (>0.5 logs) were recognized in up to 37.5% of samples. In summary, COBAS seemed to be more reliable for testing plasma viral load in HIV-infected immigrants living in Spain, one third of whom carried non-B subtypes.