AbstractThe principles governing the replication fidelity of genomes are not fully understood yet.WatsonandCrick'sbase‐pairing principle for matched deoxyribonucleotide (DNA) bases can explain why the guanine–cytosine and adenine‐thymine base pairs are approximately one hundred times more stable thermodynamically than mismatched combinations. In vitro, DNA polymerases reduce the number of mismatched base pairs to about 10−6per Watson–Crick base pair. Replication fidelity can further be enhanced to a mutation probability of 10−10or less in vivo if optimal conditions for DNA synthesis are provided by polymerase–assisting proteins and DNA‐repairing enzymes. The precise reasons for the formation of mismatched base pairs (mispairs), which are responsible for a substantial part of DNA mutations, are still in debate. Although it is agreed that a template‐directed “reading” of the hydrogen‐substitution pattern in the heterocyclic bases is crucial for proper base pairing during DNA synthesis, it is not clear which type of “misreading” leads to mispairs. Misreading may be due to a non‐Watson–Crick base pairing as well as to a change in the hydrogen‐substitution pattern, leading to Watson‐Crick‐like mispairs. The surprising discovery of the selective and quantitative DNA‐polymerase‐catalyzed formation of a pyridine‐pyrimidine base pair (involving a nucleotide base analogue) indicated that rare tautomeric forms in template DNA strands can lead to Watson‐Crick‐like mispairings that are hardly recognized by the polymerase's proofreading activity. This reveals new pathways for substitution mutations (replication‐dependent DNA point muta