The distribution of phosphofructokinase (PFK) in gray and white matter regions of the rat nervous system was evaluated. Determinations of PFK activity revealed that cell body enriched regions (sensorimotor cortex) had a significantly higher level of activity than axonal regions (sciatic nerve, dorsal roots, and optic nerve). The level of PFK activity was also significantly higher in central axons (optic nerve) than in peripheral axons (sciatic nerve). Differences in PFK activity could be largely attributed to differences in tissue content of the enzyme rather than to differences in the types of PFK isozymes present. Cortex contained significantly larger amounts of PFK relative to total protein than did peripheral nerve. However, purification of PFK revealed that all three of the PFK isozymes, C (86 kd), A (84 kd), and B (80 kd), were present in both cortex and sciatic nerve. Both SDS/PAGE and immunoblotting studies using PFK isozyme‐specific antibodies demonstrated that the relative proportions of the three PFK isozymes were similar in cell body and axonal regions of the nervous system. The PFK‐C and PFK‐A isozymes each comprised about half the total and only small amounts of the PFK‐B isozyme were present in both regions. However, immunoprecipitation experiments suggested that quantitatively different proportions of the possible PFK hybrids (tetramers) may be distributed between axonal and cell body regions. The transport of PFK was examined in this study and PFK was identified in slow component b (SCb) of axonal transport. SCb moves at a rate of 2–4 mm/day in rat axons and is known to contain several other enzymes of intermediary metabolism as well as actin. The finding that PFK, the rate limiting enzyme in glycolysis, is present in SCb lends support to the hypothesis that glycolytic enzymes are not freely diffusing proteins in axons but, instead, are present as organized assemblies that have long‐term, yet flexible, associations with structural elements of the cytoplasm.