Proteorhodopsin (PR)-containing bacteria are hypothesized to use both light and organic compounds as energy sources. Recent studies have found that PR is common in marine microorganisms, but the impact of light on the growth of PR-containing organisms and PR transcription in the environment remains unclear. We examined the diversity of PR genes and transcripts by PCR amplification and sequencing in Delaware coastal waters. Clone libraries of PR DNA and cDNA (from mRNA) revealed large differences between bacterial groups in expression of PR genes. We then evaluated by quantitative PCR the impact of light on growth and PR expression in PR-containing SAR11 bacteria (SAR11-PR) and a population of Flavobacteria (Flavobacteria-PR). This experiment was conducted in 30 l microcosms exposed to continuous light, continuous dark, and 12 h−12 h dark–light cycles for 5 days. We found a strong upregulation of PR expression by light in Flavobacteria-PR and SAR11-PR. The abundance of PR transcripts per PR cell was enhanced up to 120-fold under continuous light and up to 20-fold under dark–light cycles while continuous darkness led to very low levels of PR mRNA. This upregulation of PR expression was correlated with the abundance of PR genes, indicating net growth of SAR11-PR cells and Flavobacteria-PR under dark–light cycles. SAR11-PR and Flavobacteria-PR abundance decreased under continuous light despite upregulation of PR expression, and continuous darkness led to low abundances of both populations. Collectively, these data suggest that light affects growth of PR-containing bacteria and regulation of PR mRNA synthesis in natural communities.