The production of juvenile hormone III (JH III) by the corpora allata of the cockroach Diploptera punctata is regulated in part by peptides originating from the brain. One group of these peptides, termed allatostatins, reversibly inhibits the biosynthesis of JH in vitro. Allatostatin 4 (AST4: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is the smallest member of the AST family yet defined and was used as the benchmark peptide for these initial structure-activity studies. Two initial analog series of AST4 were examined for the ability of each analog to inhibit JH biosynthesis by corpora allata in vitro. Each analog series consisted of analogs that contained a single amino acid change from the native AST4 sequence. The first series contained Ala replacement analogs and the second contained analogs with D-amino acid replacements. The first analog series used Ala replacements to help indicate which amino acid side chains were most important for inhibition of JH biosynthesis. The most important side chain appeared to be Leu8 followed by Phe6 and Tyr4. Additionally, the D-amino acid series suggested that a secondary structural element(s) at the C-terminus of AST4 could be important to the biological activity.