Stromatolites are commonly interpreted as evidence of ancient microbial life, yet stromatolite morphogenesis is poorly understood. We apply radiometric tracer and dating techniques, molecular analyses and growth experiments to investigate siliceous stromatolite morphogenesis in Obsidian Pool Prime (OPP), a hot spring in Yellowstone National Park. We examine rates of stromatolite growth and the environmental and/or biologic conditions that affect lamination formation and preservation, both difficult features to constrain in ancient examples. The “main body” of the stromatolite is composed of finely laminated, porous, light–dark couplets of erect (surface normal) and reclining (surface parallel) silicified filamentous bacteria, interrupted by a less-distinct, well-cemented “drape” lamination. Results from dating studies indicate a growth rate of 1–5 cm year−1; however, growth is punctuated. 14C as a tracer demonstrates that stromatolite cyanobacterial communities fix CO2 derived from two sources, vent water (radiocarbon dead) and the atmosphere (modern 14C). The drape facies contained a greater proportion of atmospheric CO2 and more robust silica cementation (vs. the main body facies), which we interpret as formation when spring level was lower. Systematic changes in lamination style are likely related to environmental forcing and larger scale features (tectonic, climatic). Although the OPP stromatolites are composed of silica and most ancient forms are carbonate, their fine lamination texture requires early lithification. Without early lithification, whether silica or carbonate, it is unlikely that a finely laminated structure representing an ancient microbial mat would be preserved. In OPP, lithification on the nearly diurnal time scale is likely related to temperature control on silica solubility.