Transcription factors (TFs) interact with specific DNA regulatory sequences to control gene expression throughout myriad cellular processes. However, the DNA binding specificities of only a small fraction of TFs are sufficiently characterized to predict the sequences that they can and cannot bind. We present a maximally compact, synthetic DNA sequence design for protein binding microarray (PBM) experiments that represents all possible DNA sequence variants of a given length k (that is, all 'k-mers') on a single, universal microarray. We constructed such all k-mer microarrays covering all 10-base pair (bp) binding sites by converting high-density single-stranded oligonucleotide arrays to double-stranded (ds) DNA arrays. Using these microarrays we comprehensively determined the binding specificities over a full range of affinities for five TFs of different structural classes from yeast, worm, mouse and human. The unbiased coverage of all k-mers permits high-throughput interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution.