Phenylpropanoid polyamine conjugates have been identified in flowers of many plant species. Their presence in Arabidopsis thaliana has only been recently established in flower buds and pollen grains. Annotation and location of a cation-dependent O-methyltransferase AtTSM1 specifically to the tapetum of young flower buds enabled the subsequent identification of several genes with a putative role in phenylpropanoid polyamine conjugate biosynthesis. Based on the analysis of several A. thaliana knockout mutants, a biosynthetic pathway of these conjugates is proposed, which involves two methylation steps catalyzed by different cation-dependent O-methyltransferases, a cytochrome P450 (CYP98A8) catalyzed hydroxylation, and a conjugating acyl transfer performed by a BAHD-like, hydroxycinnamoyl (HC)-transferase. LC/MS based metabolite profiling of the cyp98A8 knockout line identified new feruloyl- and 4-coumaroylspermidine conjugates in the corresponding flowers consistent with a role of this gene in the hydroxylation of these conjugates. A pattern of minor amounts of bis- and tris-acylspermidine conjugates, likely the products of additional HC-transferases were identified in wild type as well as in the mutant lines. Transcript suppression of the genes early in the pathway was observed in knockout or RNAi-lines of the genes encoding late enzymatic steps. The implication of these findings for spermidine conjugate biosynthesis in flower buds of A. thaliana is discussed.