The use of whole-blood spots on filter paper for the detection of antibody to human immunodeficiency virus type 1 (HIV-1) was evaluated during a 20-week period under a variety of storage environments simulating the harsh tropical field conditions in Kinshasa, Zaire. During the first 6 weeks of storage, all replicates of high- and low-titer HIV-1-positive reference samples remained positive by enzyme immunoassay and Western blotting (immunoblotting), and all replicates of HIV-1-negative samples remained negative under all storage conditions. However, hot and humid storage conditions for up to 20 weeks caused a progressive decline in enzyme immunoassay optical density ratio values, which was particularly noticeable in samples with a low HIV-1 antibody titer. Harsh tropical operational conditions did not cause any repeatedly false-positive results during the 20-week storage period. The use of gas-impermeable bags with desiccant for the storage of blood spots on filter paper improved the stability of HIV-1 antibody detection over time and is recommended for the storage of whole-blood spots on filter paper in harsh tropical field settings.