The recent discovery of the aspartyl proteinase of the human immunodeficiency virus 1, (HIV-1), and its inhibition by pepstatin, has increased the search for synthetic analogues of pepstatin with increased substrate specificities. However, the therapeutic value of pepstatin is limited. For a proteinase inhibitor to have therapeutic value, it should be chemically stable, active at low concentrations, selective for a particular proteinase in the presence of other proteinases with similar substrate specificities, and should readily penetrate cell membranes. Pepstatin satisfies the first two of these criteria. The third criterion, specificity, is satisfied only at the level of the major mechanistic class. Pepstatin, while a very effective inhibitor of aspartyl proteinases, is not very selective within this class of enzymes. Pepstatin is also known not to penetrate cell membranes well due to its size and lipophilicity. In this communication we now report the preparation and determination of pepsin inhibition of eight tripeptide analogues of pepstatin. Since pepstatin is known not to penetrate cell membranes as readily as small peptides, such as leupeptin, we have synthesized tripeptide analogues of pepstatin. This was done in an effort to find a small peptide analogue with inhibitory activity comparable to that of pepstatin. The eight Cbz-protected tripeptide analogues were compared to pepstatin as inhibitors of the pepsin hydrolysis of hemoglobin. On comparing the activities, it was found that the length of the peptide chain alters the inhibitor activity. Also, the stereochemistry of carbon-3, bearing the hydroxyl group, is very important to the activity of the inhibitors. The 3S,4S diastereomers were in each case shown to be better inhibitors of the peptic hydrolysis of hemoglobin than the 3R,4S diastereomers. Modifications made in the side chain of the novel amino acid statine had the greatest effect on the pepsin inhibition. Analogues containing a statine unit derived from L-phenylalanine (6a,b) were significantly less active as pepsin inhibitors, and very little inhibition of pepsin activity is shown by the analogues containing a statine unit derived from L-tyrosine (7a,b), indicative of steric restrictions within the enzyme pepsin. Interestingly, analogues containing a statine unit derived from L-norleucine (8a,b) displayed better inhibition of pepsin activity than those containing statine units derived from L-leucine. In view of the preliminary success achieved in finding a small peptide with potency similar to pepstatin for pepsin inhibition, further modifications to improve hydrophilicity of aspartyl proteinase inhibitors are warranted. Further synthesis and testing of tripeptide analogues of pepstatin is currently underway.