The use of acridine orange to visualize and quantify spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm was investigated. Bacterial colonies supported on polycarbonate filter membranes were grown on R2A agar plates. Some colonies were sampled for cell enumeration, while others were cryoembedded, sectioned, and stained with the fluorescent nucleic acid stain acridine orange. Spatial patterns of fluorescent color and intensity with depth in the colony were quantified using confocal microscopy and image analysis of stained cross sections. Colonies sampled in the midexponential phase were thin (20 microns), had high average specific growth rates (> 1 h-1), and had all the cells stained bright orange. Colonies sampled after more than 24 h of growth were thick (> 200 microns) and were growing slowly (mu < 0.15 h-1). These older colonies were characterized by distinct bands of orange at the colony edges and a dark green center. Stained biofilm cross sections displayed a similar orange band at the biofilm-bulk fluid interface and a green interior. Colony-average specific growth rates, determined by calculating the local slope of the cell accumulation versus time data, were correlated with colony-average fluorescence intensities. There was no correlation between average specific growth rate and orange or green intensity individually, but growth rate did correlate with the orange:green intensity ratio (r2 = 0.57). The resulting regression was used to predict specific growth rate profiles within colonies. These profiles indicated that bacteria were growing rapidly near the air and agar interfaces and more slowly in the center of the colonies when thicker than about 30 microns. The dimension of the orange bands ranged from 10 to 30 microns, which may indicate the thickness of growing regions. The inherent variability associated with this technique suggests that it is best applied in single species systems and that the results should be regarded as qualitative in nature.