Background Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis. Results Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative ?-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active ?-1,3 endoglucanase. Conclusion Time-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding ?-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides