In nature, bacteria are able to form complex surface-attached communities called biofilms. Microbial biofilms pose a particular problem in many human infections because of an inherent tolerance to antimicrobial agents and host immune killing and clearance. We have used complementary DNA (cDNA) microarray technology to identify Pseudomonas aeruginosa genes that are differentially expressed in growing and developing biofilms. Our study shows that, when compared with planktonic bacteria, gene expression profiles of biofilm cells have the highest resemblance to the profiles of stationary-phase cells. We suggest that the process of biofilm development involves a series of adaptive responses including those to anaerobic and iron-limitation stresses, rather than being associated with a unique biofilm developmental program. Mapping of quorum-sensing regulated genes in a P. aeruginosa biofilm identified a set of N-acyl homoserine lactone (AHL)-dependent genes that are exclusively expressed in sessile cells. One of these genes, pvdQ, encodes an AHL acylase that degrades long-acyl but not short-acyl AHLs. This result may provide an explanation for the previous finding that the level of long-acyl AHLs is greatly reduced in P. aeruginosa biofilm cells as compared with their planktonic counterparts. Furthermore, we present evidence that quorum sensing is participating in the control of iron-limitation responses in the biofilm cells.