Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (alpha(2)) and R2 (beta(2)), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.