Naegleria actin retains conserved binding sites for small-molecule inhibitors of actin dynamics. A multiple sequence alignment was generated to compare N. gruberi’s (N.g.’s) most highly expressed actin protein sequence with that of other eukaryotic actins (see Table S3 for sequences and ID numbers). This alignment includes actin sequences from other Discobids, N. fowleri (N.f.) and Trypanosoma cruzi (T.c.); Metamonads, Giardia intestinalis (G.i.) and Trichomonas vaginalis (T.v.); Plants, Chlamydomonas reinhardtii (C.r., including NAP1 [N] and IDA5 [I]) and Ectocarpus siliculosus (E.s.); Stramenopiles, Thalassiosira pseudonana (T.p.); Alveolates, Toxoplasma gondii (T.g.), Plasmodium falciparum (P.f., including actin-1 and actin-2), and Tetrahymena thermophila (T.t.); Amoebozoa, Dictyostelium discoideum (D.d.), Entamoeba histolytica (E.h.), and Acanthamoeba castellanii (A.c.); and Opisthokonts, Saccharomyces cerevisiae (S.c.), Schizosaccharomyces pombe (S.p.), and Oryctolagus cuniculus (O.c.). Sequences were aligned using T-Coffee (Notredame et al., 2000) with defaults (Blosum62 matrix, gap open penalty = −50, gap extension penalty = 0) in Jalview, and residues were colored based on conservation (grayscale) or to highlight differences in amino acids within drug-binding sites (ILVAM: salmon; FWY: tangerine; KRH: blue; DE: red; STNQ: lime; PG: raspberry; and C: yellow). Bars indicate important residues for drug binding, and binding sites are abbreviated as follows: L, latrunculin; P, phalloidin; C, cytochalasin; J, jasplakinolide.