The extracellular biofilm matrix includes primarily DNA and exopolysaccharides (EPS), which function to maintain aggregate structures and to protect biofilms from antibiotics and the immune response. Both polymers are anionic and have cation binding activity, however the impact of this activity on biofilms is not fully understood. Host cell contact is considered the primary signal for activation of most type III secretion systems (T3SS), although calcium limitation is frequently used as a trigger of contact-independent T3SS expression. We hypothesized that alginate, which is a known calcium binding exopolysaccharide produced in mucoid Pseudomonas aeruginosa isolates, can activate the T3SS in biofilms. The addition of exogenous purified alginate to planktonic, non-mucoid PAO1 cultures induced expression of exoS, exoT and exoY-lux reporters of the T3SS in a concentration-dependent manner. Induction by alginate was comparable to induction by the calcium chelator NTA. We extended our analysis of the T3SS in flow chamber-cultivated biofilms, and showed that hyperproduction of alginate in mucA22 mucoid isolates resulted in induction of the exoS-gfp transcriptional reporter compared to non-mucoid paired isolates. We confirmed the transcriptional effects of alginate on the T3SS expression using a FlAsH fluorescence method and showed high levels of the ExoT-Cys(4) protein in mucoid biofilms. Induction of the T3SS could be prevented in planktonic cultures and mucoid biofilms treated with excess calcium, indicating that Ca(2+) chelation by the EPS matrix caused contact-independent induction. However, mucoid isolates generally had reduced exoS-lux expression in comparison to paired, non-mucoid isolates when grown as planktonic cultures and agar colonies. In summary, we have shown a mucoid biofilm-specific induction of the type III secretion system and highlight a difference between planktonic and biofilm cultures in the production of virulence factors.