The contents and molecular forms of five different prodynorphin-derived opioid peptides were compared in extracts of rat hippocampus by radioimmunoassay after C18-HPLC resolution. Dynorphin (Dyn) A(1-17) immunoreactivity (ir) and Dyn B-ir were heterogeneous in form; Dyn A(1-8)-ir, alpha-neoendorphin (alpha neo)-ir and beta-neoendorphin (beta neo)-ir each eluted as single homogeneous peaks of immunoreactivity. The fraction of immunoreactivity having the same retention as the appropriate synthetic standard was used to estimate the actual hippocampal content of each peptide. Comparison of these values showed that the concentrations of Dyn B, alpha neo, and Dyn A(1-8) were nearly equal, whereas both Dyn A(1-17) and beta neo were 1/5th to 1/10th the value of the other three. Calcium-dependent K+-stimulated release of these prodynorphin-derived opioids from hippocampal slices was detected. The stimulated rates of release were highest for Dyn B-ir followed by alpha neo-ir, then beta neo-ir and Dyn A(1-8)-ir with Dyn A(1-17)-ir lowest. The relative rates of stimulated release were in agreement with the relative proportions of peptide present within the tissue. This evidence of the presence and release of these opioid peptides considerably strengthens the hypothesis that this family of endogenous opioids plays a neurotransmitter role in the hippocampus.