To determine if unconventional myosins play a role in nerve outgrowth, antibodies specific for rat brain derived mammalian myosin I alpha (MMI alpha) were used to label cultured rat superior cervical ganglion nerve cells. Observations were made at both the light and electron microscopic level of resolution using preparative procedures designed to enhance the ability to precisely determine the relationship between antibody label and cellular structures in order to map the distribution and structural association of this myosin. Immunofluorescence showed that MMI alpha has a punctate distribution throughout the nerve cell body, neurites, and growth cones. In growth cones, MMI alpha staining is sometimes elevated in thin peripheral regions of high actin content at the leading edge. Immunoelectron microscopy using colloidal gold conjugated antibodies showed that in growth cones MMI alpha is absent from membranous organelles and is concentrated primarily in the cell cortex adjacent to the cell membrane. The cortical label is equally distributed between upper and lower membranes. The plasma membrane association of the MMI alpha label persists under conditions in which the actin cytoskeleton is perturbed or removed, suggesting a direct association between a fraction of MMI alpha and the plasma membrane. MMI alpha label is also associated with the non-cortical actin cytoskeleton. Partial disruption of the actin cytoskeleton using cytochalasin B causes redistribution of only a subset of MMI alpha label. These data suggest a complex relationship between MMI alpha, the actin cytoskeleton, and the plasma membrane in the growth cone. In contrast to its localization in the growth cone, in neuronal cell bodies MMI alpha is also associated with tubulovesicular structures. This suggests that at this location MMI alpha may either act as an organelle motor or is passively transported to the plasma membrane on vesicles.