Actinorhodin production is markedly enhanced when an acidic pH shock is applied to a surface-grown culture of Streptomyces coelicolor A3(2). For an in-depth study of this phenomenon, transcriptional analyses using DNA microarrays and reverse transcription polymerase chain reaction and proteomic analysis were performed. Investigated were expression levels of the regulators and enzymes responsible for signal transduction and actinorhodin biosynthesis and enzymes involved in some major metabolic pathways. Regulators PkaG, AfsR, AfsS and/or another unidentified regulator and ActII-ORF4, in sequence, were observed to be activated by pH shock. In addition, a number of genes associated with actinorhodin production and secretion and the major central metabolic pathways investigated were observed to be upregulated with pH shock. Fatty acid degradation was particularly promoted by pH shock, while fatty acid biosynthesis was suppressed; it is envisaged that this enriches the precursor pool (acetyl-CoA) and building blocks for actinorhodin biosynthesis. Furthermore, glucose 6-phosphate dehydrogenases, initiating the pentose phosphate pathway, were highly activated by pH shock, enriching the reduced nicotinamide adenine dinucleotide phosphate (NADPH) pool for biosynthesis in general. It is deduced that these metabolic changes caused by pH shock have positively contributed to the stimulation of actinorhodin biosynthesis in a concerted manner.