- Ultramafic rocks are exposed at the core of a domal structure near a fossil ridge–transform intersection (RTI) in the Troodos ophiolite. A sequence of heavily serpentinized rocks occurs adjacent to a major axis–parallel fault, the Amiandos Fault (AF), which juxtaposes them against gabbro. Previously, serpentinization and faulting were not considered to be related to the Cretaceous ocean spreading history of the Troodos ophiolite, but instead were interpreted as associated with late, emplacement-related tectonics and diapirism. Unusually high δ18O values previously measured in Troodos serpentine (Srp) supported this view. Here, petrographic and isotope tracers (δD, δ18O) of water–rock interaction are examined in three profiles across the AF to determine the spatial distribution, temperature, and the type of water involved in serpentinization in the Troodos RTI. Complete serpentinization, widespread chrysotile veining and very high δ18O (Srp), 10.6 to 12.6‰, characterize the rocks along a 2.5 km long profile at the central part of the serpentinite exposure. Much lower δ18O(Srp) values, 4.6 to 6.6‰, were measured at the AF fault zone in the highly serpentinized 300 m–long northern profile. Hydrogen isotope ratios also spatially vary: from − 70 to − 86‰ and − 57 to − 75‰ in the central and northern profiles, respectively. The bimodal distribution of isotope ratios calls for two distinct serpentinization events: localized ‘oceanic-type’ hydrothermal (100–200 °C) alteration initiated by deep infiltration of seawater during seafloor spreading followed by pervasive ‘ophiolite-type’ low-temperature hydration and veining along the AF. Superposition of the two serpentinization events is evident at a third, 2 km long southernmost profile, where δ18O (Srp) values decrease gradually from 10.7‰ 2 km away from the fault to 5.0‰ at the AF fault zone. Post-magmatic decrease of δ18O(plagioclase) in gabbros in the footwall of the AF and the apparent lack of amphibolitization suggest high-temperature, off-axis gabbro-water interaction and focused fluid flow to the lower crust through the AF zone. These observations suggest that the Amiandos fault was active during seafloor spreading, possibly operating as a detachment fault in a core complex structure, exhuming progressively deeper levels of the oceanic lithosphere. This scenario is supported by additional observations such as the proximity to RTI and association with highly-rotated blocks in the sheeted dikes.