Lamination in stromatolites (considered some of the oldest fossils on Earth) is commonly interpreted to record the periodic response of a microbial community to daily, seasonal, or perhaps yearly environmental forcing, but the inability to date ancient stromatolites precludes an understanding of the lamination formation processes. We use high-resolution 14C dating of Holocene stromatolites from Walker Lake, Nevada (United States), to construct a record of lamination rate over the course of accretion. Laminae formed with a period of 5.6 ± 1.6 yr/lamination at the base of the structure, 1.6–2.8 ± 1.9 yr/lamination in the middle, and 4.5 ± 0.8 yr/lamination at the top of the laminated portion. The predominant 4−6 yr periodicity indicates that lamination formation is likely more closely related to regional climate forcing (e.g., El Niño–Southern Oscillation) versus the typical diurnal or seasonal changes in microbial mats traditionally assumed for most ancient stromatolites. Thus, generalizations regarding the influence of microbial mats on stromatolite lamination and the use of stromatolites as biosignatures need careful consideration.