The ability to reproducibly colonize substrata with concomitant monitoring of biofilm development is essential to laboratory studies of microbial activities at surfaces. A test system was developed whereby on‐line, non‐destructive measurements (open circuit potential [OCP], direct cell counts, bioluminescence, oxygen) of bacterial colonization and metabolic activity could be obtained from a series of laminar‐flow adhesion cells. The cells consisted of two high‐density polyethylene blocks 32 mm H × 65 mm W × 178 mm L, with a 1 mm deep flow channel milled in the top block. A glass viewing window enabled direct observation of a removable, flush‐mounted 25 x 50 mm coupon, which was recessed into the bottom block. Laminar‐flow conditions were validated at linear flow rates up to 1.3 cm s−1(20 ml min−1). Reproducible colonization ofPseudomonas fluorescensmonocultures was obtained, with 72 h direct‐counts and viable counts ranging from between 5.1 to 10.4 × 107and 1.4 to 2.2 × 107cells cm−2, respectively, for replicated flow cells (n=20). In situpulse‐labeling of intact biofilms with14C‐acetate resulted in reproducible incorporation of the radiolabel into cell membrane lipids, with 60 min uptake values ranging from 1.0 to 1.8 × 10−5DPM cell−1. On‐line OCP measurements remained stable in sterile test systems, varying by less than 12 mV, for over 80 h. The flow cells provided a means for reproducibly colonizing various substrata underin situenvironmental conditions without perturbing the developing biofilms.