The identification of genetic factors predisposing or protecting against HIV-1 infection has been an important aim in AIDS research. Two of these factors are located in the promoter region of the CCL5 gene, which encodes the RANTES (regulated on activation, normal T cells expressed and secreted) chemokine, an inhibitor agent for M-tropic HIV-1 strains. More specifically, the role of single-nucleotide polymorphisms (SNPs) -403G --> A and -28C --> G has been evaluated in the course of HIV-1 infection in several populations with different genetic, geographic, and ethnic backgrounds. Here we present a fast, simple, reliable, and efficient method for the simultaneous genotyping of these two CCL5 variants. A case-control study has been performed to evaluate the role of -403G --> A and -28C --> G as susceptibility factors for HIV-1 infection in the Spanish population. No differences have been found in the allelic frequencies of either variant or in the haplotype/genotype distribution between patients and controls. These data would be consistent with a lack of association between these SNPs and HIV-1 infection in our population.