We have isolated a number of clones believed to contain the origins of DNA replication in monkey cells. A detailed analysis of nine of these has been completed. Only two contain unique sequences. The remaining seven contain moderately reiterated or highly reiterated sequences. One moderately reiterated monkey sequence of 196 bp (base pair) has been found in two different clones. All of the sequences contain extensive AT-rich regions and potential stem-loop structures. Six of the nine share between 13 and 19 bp of a 21 bp "consensus sequence." Four of five tested replicate preferentially early in S phase. None has been shown to act as a plasmid replication origin. In other experiments, we have generated deletions and substitutions of the 27 bp perfect inverted repeat sequence (13x1x13) at the SV40 replication origin. A substitution mutant, in which an entirely different 13x13 perfect repeat has been substituted for the original 13x1x13 sequence, is incapable of binding the SV40 T antigen. Heteroduplex molecules between the wild type and mutant molecule obligatorily exist as cruciform structures, and on reconstitution with histones cause nucleosome phasing.