We report here the application of biocytin (a biotin-lysine complex) as an extracellular tracer in vitro. Biocytin was applied extracellularly, revealing Golgi-like staining of cells in the adult in vitro rat visual cortex. Micropipettes were filled with a solution of 2.3-2.6% biocytin dissolved in 0.05 M Tris buffer, pH 7.4. Biocytin was applied by one of 3 methods: diffusion, pressure injection or drop application. Cell bodies and dendrites around the application site and their efferent axonal processes were stained; dendritic spines were often visible. The injection sites varied in size from a single cell to a diameter of 400 microns. When applied in layer I-III, few filled cells were also seen in layers IV and V, outside the application site. The drop application (5-10 microliters) of biocytin resulted in filling of cells throughout the cortex. The combination of biocytin and the slice preparation was found to be very useful in revealing cell morphology and tracing interlaminar connections in the visual cortex. The advantages of this technique are its ease of application, the precise and restricted injection sites, and Golgi-like morphological detail.