To gain insight on the significance of human T-cell lymphotropic virus type 1 (HTLV-1) indeterminate serological reactivities, we studied villagers of South Cameroon, focusing on a frequent and specific HTLV-1 Gag indeterminate profile (HGIP) pattern (gag p19, p26, p28, and p30 without p24 or Env gp21 and gp46). Among the 102 sera studied, 29 from all age groups had a stable HGIP pattern over a period of 4 years. There was no epidemiological evidence for sexual or vertical transmission of HGIP. Seventy-five percent of HGIP sera reacted positively on MT2 HTLV-1-infected cells by immunofluorescence assay. However, we could not isolate any HTLV-1 virus or detect the presence of p19 Gag protein in cultures of peripheral blood mononuclear cells obtained from individuals with strong HGIP reactivity. PCR experiments conducted with primers for HTLV-1 and HTLV-2 (HTLV-1/2 primers) encompassing different regions of the virus did not yield HTLV-1/2 proviral sequences from individuals with HGIP. Using 11 peptides corresponding to HTLV-1 or HTLV-2 immunodominant B epitopes in an enzyme-linked immunosorbent assay, one epitope corresponding to the Gag p19 carboxyl terminus was identified in 75% of HGIP sera, while it was recognized by only 41% of confirmed HTLV-1-positive sera. A positive correlation between HTLV-1 optical density values and titers of antibody to Plasmodium falciparum was also demonstrated. Finally, passage of sera through a P. falciparum-infected erythrocyte-coupled column was shown to specifically abrogate HGIP reactivity but not the HTLV-1 pattern, suggesting the existence of cross-reactivity between HTLV-1 Gag proteins and malaria-derived antigens. These data suggest that in Central Africa, this frequent and specific Western blot is not caused by HTLV-1 infection but could instead be associated with P. falciparum infection.