Carbonate deposits in Roman aqueducts of Patara and Aspendos (southern Turkey) were studied to analyse the nature of their regular layering. Optical microscopy and electron-backscattered diffraction results show an alternation of dense, coarsely crystalline, translucent laminae composed of bundles and fans of elongate calcite crystals with their c-axes parallel to the long axis, and porous, fine-grained laminae with crystals at near-random orientation. The δ18O and δ13C data show a strong cyclicity and anti-correlation, whereby high and low δ18O values correspond to dense columnar and porous fine-grained laminae, respectively. Geochemical analyses show similar cyclic changes in carbonate composition. Electron microprobe and laser ablation inductively coupled mass spectrometry analyses show that porous fine-grained laminae are enriched in elements associated with detrital material (Fe, Mg, K, Al and Si), whereas the dense columnar laminae are nearly pure calcite. The gradient in major and trace-element distribution, regular changes in crystal type and in oxygen and carbon-isotope composition from porous fine-grained to dense columnar laminae reflect changes in water chemistry, discharge, temperature and biological activity. Because of the strong bimodal cyclicity of the Mediterranean climate in southern Turkey, the observed laminae can be attributed to calcite deposition during the dry (porous fine-grained) and wet season (dense columnar), respectively. This observation implies that, with proper geochemical and microstructural control, lamination in carbonate deposits in Roman aqueducts can be used for relative dating of aqueduct construction and maintenance and to obtain data on external factors that influenced the aqueducts, such as palaeoclimate and natural hazards. Carbonate deposits in Roman aqueducts show properties of both flowstone speleothems and riverine fresh water tufa. As many aqueducts of nearly identical channel geometry are present in different climate zones and with different source water characteristics, they can be used as natural experiment setups to test and improve existing models of how laminated fresh water carbonates record climate on time scales ranging from seasonal to millennial.