The relative abundance of transcripts encoding proteins involved in inorganic carbon concentrating mechanisms (CCM), detoxification of reactive oxygen species (ROS) and photosynthesis in the thermophilic cyanobacterium Synechococcus OS-B′ was measured in hot spring microbial mats over two diel cycles, and was coupled with in situ determinations of incoming irradiance and microenvironmental dynamics of O₂ and pH. Fluctuations in pH and O₂ in the mats were largely driven by the diel cycle of solar irradiance, with a pH variation from ∼7.0 to ∼9.5, and O₂ levels ranging from anoxia to supersaturation during night and day, respectively. Levels of various transcripts from mat cyanobacteria revealed several patterns that correlated with incident irradiance, O₂ and pH within the mat matrix. Transcript abundances for most genes increased during the morning dark–light transition. Some transcripts remained at a near constant level throughout the light period, whereas others showed an additional increase in abundance as the mat underwent transition from low-to-high light (potentially reflecting changes in O₂ concentration and pH), followed by either a decreased abundance in the early afternoon, or a gradual decline during the early afternoon and into the evening. One specific transcipt, psbA1, was the lowest during mid-day under high irradiance and increased when the light levels declined. We discuss these complex in situ transcriptional patterns with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle.