Corn gluten meal (CGM), a by‐product of corn (Zea mays L.) wet‐milling, has been shown to be an effective, natural preemergence herbicide and fertilizer for various plant production systems. Alaninylalanine (Ala‐Ala), along with four other dipeptides were isolated from hydrolyzed CGM and identified as being the inhibitory compounds. The herbicidal effects of these natural compounds are seen as growth‐regulating root inhibitors that have minimal effect on shoot growth at low concentrations. However, little is known about the precise inhibitory action of CGM or Ala‐Ala. The objective of this research was to elucidate morphological and anatomical differences in perennial ryegrass (Lolium perenne L.) seedlings treated with Ala‐Ala by means of light and transmission electron microscopy, as well as autoradiographic studies using [3H+]‐Ala‐Ala. Root length was reduced by at least 42% at all treatment concentrations when compared with the control. The treated root tips were observed to be devoid of cellular components, specifically discernible nuclei and mitotic structures, with an overall loss of cytoplasmic integrity. Treated root tips had cell wall abnormalities, including uneven thickening and breakage, especially in the epidermal and the adjacent cortical cells. Autoradiographs suggested that, at treatment concentrations causing epidermal damage, there was minimal inward movement of the dipeptide. At low treatment concentrations, root tip epidermal necrosis was not evident, and inward movement of the dipeptide was not impeded. The herbicidal effects of the dipeptide, Ala‐Ala, on treated perennial ryegrass roots were numerous. Higher concentration levels seemed to cause more of an epidermal necrosis, whereas, lower concentrations produced more subtle effects to the cellular processes.