Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes a protein of unknown function. Complementation analysis and phage-mediated transduction demonstrated that the transposon insertion in PA5346 was the cause of the biofilm-defective phenotype. Examination of flow cell-grown biofilms showed that the sadB199 mutant could initiate surface attachment but failed to form microcolonies despite being proficient in both twitching and swimming motility. Closer examination of early attachment revealed an increased number of the sadB199 mutant cells arrested at reversible attachment, functionally defined as adherence via the cell pole. A positive correlation among biofilm formation, irreversible attachment, and SadB level was demonstrated, and furthermore, RpoN and FleR appear to negatively affect SadB levels. Fractionation studies showed that the SadB protein is localized to the cytoplasm, and with the use of GPS-linker scanning mutagenesis, the C-terminal portion of SadB was shown to be dispensable for function, whereas the two putative domains of unknown function and the linker region spanning these domains were required for function. We discuss the results presented here in the context of microbial development as it applies to biofilm formation.