Protein entry into the secretory pathway is initiated by translocation into the endoplasmic reticulum. ER signal peptides direct protein import into the organelle. Signal peptide (h-region) variants of a variant surface glycoprotein, VSG-117, and small molecule inhibitors were used to determine the properties of trypanosome signal peptides and factors affecting protein translocation into the ER of T. brucei. h-Regions of signal peptides are required for signal peptide activity. h-Region peak hydrophobicity is thought to be crucial to signal peptide activity. We find that peak hydrophobicity alone does not confer signal peptide activity, and that T. brucei h-regions possess tri-component peptide motifs (e.g. L-L-x-[AILV], L-x(1,2)-L-[AILV], and L-x(2,3)-L-[AILPV]). The h-motifs have flexible amino acid requirements, and function with either hydrophobic or serine residues. The order of amino acids in h-motifs is essential to signal peptide activity. Import of VSG into the ER enables trafficking of the protein to the plasma membrane, and is important for T. brucei viability. In hopes of finding new lead candidates for new anti-trypanosome drug discovery we sought to identify compounds that blocked VSG import into T. brucei ER microsomes. Sodium azide, but not valinomycin, inhibited VSG-117 translocation into TbRM, suggesting that an azide-sensitive factor is important for protein translocation into TbRM. We identified pyrimidinone-peptoids and cyclopeptolides as two classes of compounds that block VSG-117 translocation into TbRM, and were found to be trypanocidal. Specifically, both MAL3-101 and NFI028 blocked ER protein import and killed T. brucei (IG50 of 1.5 M for each). We propose pyrimidinone-peptoid and cyclopeptolide derivatives as lead compounds for discovery of new anti-trypanosome drugs.