Human alpha-synuclein (alpha S) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of alpha S into amyloid fibrils. Using bacterially expressed acetylated-alpha S (NTAc-alpha S) and endogenous alpha S (Endo-alpha S) from human erythrocytes, we show that N-terminal acetylation has little impact on alpha S binding to anionic membranes and thus likely not relevant for regulating membrane affinity. N-terminal acetylation does have an effect on alpha S aggregation, resulting in a narrower distribution of the aggregation lag times and rates. 2D-IR spectra show that acetylation changes the secondary structure of alpha S in fibrils. This difference may arise from the slightly higher helical propensity of acetylated-alpha S in solution leading to a more homogenous fibril population with different fibril structure than non-acetylated alpha S. We speculate that N-terminal acetylation imposes conformational restraints on N-terminal residues in alpha S, thus predisposing alpha S toward specific interactions with other binding partners or alternatively decrease nonspecific interactions.