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Abstract Early diagenetic dolomite beds were sampled during the Ocean Drilling Programme (ODP) Leg 201 at four reoccupied ODP Leg 112 sites on the Peru continental margin (Sites 1227/684, 1228/680, 1229/681 and 1230/685) and analysed for petrography, mineralogy, δ 13 C, δ 18 O and 87 Sr/ 86 Sr values. The results are compared with the chemistry, and δ 13 C and 87 Sr/ 86 Sr values of the associated porewater. Petrographic relationships indicate that dolomite forms as a primary precipitate in porous diatom ooze and siliciclastic sediment and is not replacing the small amounts of precursor carbonate. Dolomite precipitation often pre‐dates the formation of framboidal pyrite. Most dolomite layers show 87 Sr/ 86 Sr‐ratios similar to the composition of Quaternary seawater and do not indicate a contribution from the hypersaline brine, which is present at a greater burial depth. Also, the δ 13 C values of the dolomite are not in equilibrium with the δ 13 C values of the dissolved inorganic carbon in the associated modern porewater. Both petrography and 87 Sr/ 86 Sr ratios suggest a shallow depth of dolomite formation in the uppermost sediment (<30 m below the seafloor). A significant depletion in the dissolved Mg and Ca in the porewater constrains the present site of dolomite precipitation, which co‐occurs with a sharp increase in alkalinity and microbial cell concentration at the sulphate–methane interface. It has been hypothesized that microbial ‘hot‐spots’, such as the sulphate–methane interface, may act as focused sites of dolomite precipitation. Varying δ 13 C values from −15‰ to +15‰ for the dolomite are consistent with precipitation at a dynamic sulphate–methane interface, where δ 13 C of the dissolved inorganic carbon would likewise be variable. A dynamic deep biosphere with upward and downward migration of the sulphate–methane interface can be simulated using a simple numerical diffusion model for sulphate concentration in a sedimentary sequence with variable input of organic matter. Thus, the study of dolomite layers in ancient organic carbon‐rich sedimentary sequences can provide a useful window into the palaeo‐dynamics of the deep biosphere.