Phytohormone overproduction in crown gall tumors is due to the expression of several T-DNA genes. The data strongly suggest that the tmr gene (transcript 4) is responsible for cytokinin overproduction by encoding dimethylallyl-pyrophosphate:AMP dimethylallyltransferase (DMA transferase), an enzyme directly involved in cytokinin biosynthesis. Cell-free extracts of Escherichia coli strains containing the tmr gene from pTiA6NC had DMA transferase activity. No activity was present in the control strain containing only the plasmid vector. The cytokinins synthesized were isopentenyladenine, isopentenyladenosine, and isopentenyladenosine 5'-monophosphate. DMA transferase activity was also detected in cloned crown gall tumors incited by Agrobacterium tumefaciens wild-type A6NC and a tms mutant. Enzymatic activity in cell-free extracts of E. coli and tumors could be abolished by transposon insertion within the tmr gene.