Capsaicin is a bioactive molecule synthesized by enzymatic (putative capsaicin synthase) condensation of vanillylamine, a phenyl propanoid intermediate with 8-methyl-nonenoic acid, a fatty acid derivative from leucine/valine pathway. Analysis of levels of 8-methyl-nonenoic acid and phenyl propanoid intermediates in high, medium, and low pungent Capsicum genotypes revealed that the 8-methyl-nonenoic acid pool plays a crucial role in determining the efficacy of capsaicin levels. Cerulenin-mediated inhibition of 8-methyl-nonenoic acid synthesis decreased the capsaicin biosynthesis in Capsicum cell suspension cultures. Similarly amino oxy acetate inhibited vanillylamine synthesis but failed to reduce capsaicin production. The mRNA transcript analysis of keto acyl synthase (KAS), a crucial enzyme involved in 8-methyl-nonenoic acid and an amino transferase (AMT), involved in vanillylamine biosynthesis was studied. The mRNA transcript analysis revealed the progressive developmental expression of KAS gene in the placenta during the ontogeny of the fruit, whereas AMT transcripts levels did not show significant differences. Hence, the study demonstrates the influence of 8-methyl-nonenoic acid and its possible regulatory role in capsaicin biosynthesis in Capsicum spp.