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3(R)-Hydroxy-4(R)-methyl-6(E)-octenoic acid, the C9-backbone of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (Bmt), is biosynthesized as a coenzyme A thioester from acetyl-CoA, malonyl-CoA, NADPH, and S-adenosylmethionine via a polyketide pathway. Here we present detailed enzymatic studies about the basic assembly process. After attachment of the activated building units to Bmt polyketide synthase the intermediates remained enzyme-bound throughout the cycle. Premature cutoff of biosynthesis led to the release of the intermediates from the enzyme, either as coenzyme A thioesters or, in the case of reactive C8-intermediates, as lactones. Enzyme-bound 3-oxo-4-hexenoic acid, the condensation product of the second elongation cycle, could be identified as the exclusive substrate for the introduction of the methyl group. Part of the biosynthesis including the first elongation cycle, the second condensation reaction, and the methylation step was shown to follow a processive mechanism. All activated intermediates of this processive part could be introduced into the correct pathway at the respective steps, whereas 2-methyl-3-oxo-4-hexenoyl-CoA and all following methylated intermediates were not able to enter the cycle any more. Obviously, the region of Bmt polyketide synthase responsible for this latter part of the biosynthetic pathway is inaccessible for externally supplied coenzyme A thioesters. Butyryl-CoA was recognized by Bmt polyketide synthase with an efficiency comparable to that of crotonyl-CoA and processed to 3-hydroxy-4-methyloctanoyl-CoA, the saturated analog of the natural basic assembly product, indicating a relaxed specificity of Bmt polyketide synthase with respect to the starter unit.