The mammalian transcription factor SRF and the yeast regulatory protein MCM1 contain DNA binding domains that are 70% identical; moreover, both proteins can bind the serum response element in the human c-fos promoter. Here we present an analysis of MCM1 sequence specificity by selection of sites from random sequence oligonucleotides. In this assay the MCM1 DNA binding domain selects binding sites containing the consensus (NotC)CCY(A/T)(A/T)(T/A)NN(A/G)G, distinct from the SRF binding consensus CC(A/T)6GG. Carboxylethylation interference analysis of a set of selected sites suggests that MCM1 contacts DNA in its major groove throughout one helical turn. These differences in specificity are largely due to sequence differences between the N terminal basic parts of the SRF and MCM1 DNA binding domains. Comparison of the relative binding affinities of MCM1 and SRF for a panel of representative binding sites showed that many high affinity MCM1 sites have negligible affinity for SRF and vice versa. Thus MCM1 and SRF have significantly different sequence specificities.