Despite major advancements in development of antiretroviral therapy, currently there is no effective treatment to block the impact of HIV-Tat protein which is released extracellularly and can cause activation of lymphocytes, glial cells and neurotoxicity. Because the structure of extracellular Tat is currently unknown, we used atomic force microscopy, circular dichroism and computer simulation to study the structure and aggregation characteristics of HIV-Tat protein. We found that about 8% of the HIV-Tat population was in monomeric state, the rest being in aggregated state. The aggregates ranged in size from dimers, trimers, tetramers to large oligomers (50-mers and larger) and all structures presented a globular shape. Circular dichroism measurements suggested that 20% of the sample's structure is alpha-helical, which likely represents the aggregated state, since monomers were few. The reducing agent dithiothreitol broke down the large aggregates, leading to a population of smaller aggregates, but structures smaller than the monomer were found as well, indicating that the molecule is prone to rupture under reducing conditions. CuSO4 induced dimerization and larger aggregates of HIV-Tat. A change of 14-19 aminoacids in protein structure, including the Cys 31 replacement with Ser led to a larger number of monomers in the sample (11%), a distribution of smaller aggregates, and reduced adherence to mica. Freezing of Tat reduced the aggregates size, increased their adherence capacity and ruptured the molecule. Further studies will investigate the effect of protein concentration, solution ionic strength, temperature variation and time of storage in solution, to characterize the aggregated state and its reversibility.