BACKGROUND: Metagenomics enables the analysis of bacterial population composition and the study of emergent population features, such as shared metabolic pathways. Recently, we have shown that metagenomics datasets can be leveraged to characterize population-wide transcriptional regulatory networks, or meta-regulons, providing insights into how bacterial populations respond collectively to specific triggers. Here we formalize a Bayesian inference framework to analyze the composition of transcriptional regulatory networks in metagenomes by determining the probability of regulation of orthologous gene sequences. We assess the performance of this approach on synthetic datasets and we validate it by analyzing the copper-homeostasis network of Firmicutes species in the human gut microbiome. RESULTS: Assessment on synthetic datasets shows that our method provides a robust and interpretable metric for assessing putative regulation by a transcription factor on sets of promoter sequences mapping to an orthologous gene cluster. The inference framework integrates the regulatory contribution of secondary sites and can discern false positives arising from multiple instances of a clonal sequence. Posterior probabilities for orthologous gene clusters decline sharply when less than 20 % of mapped promoters have binding sites, but we introduce a sensitivity adjustment procedure to speed up computation that enhances regulation assessment in heterogeneous ortholog clusters. Analysis of the copper-homeostasis regulon governed by CsoR in the human gut microbiome Firmicutes reveals that CsoR controls itself and copper-translocating P-type ATPases, but not CopZ-type copper chaperones. Our analysis also indicates that CsoR frequently targets promoters with dual CsoR-binding sites, suggesting that it exploits higher-order binding conformations to fine-tune its activity. CONCLUSIONS: We introduce and validate a method for the analysis of transcriptional regulatory networks from metagenomic data that enables inference of meta-regulons in a systematic and interpretable way. Validation of this method on the CsoR meta-regulon of gut microbiome Firmicutes illustrates the usefulness of the approach, revealing novel properties of the copper-homeostasis network in poorly characterized bacterial species and putting forward evidence of new mechanisms of DNA binding for this transcriptional regulator. Our approach will enable the comparative analysis of regulatory networks across metagenomes, yielding novel insights into the evolution of transcriptional regulatory networks.