Western blotting of low molecular weight ( M r) acidic and basic isoelectric point (p I) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, M r 6045, p I 4.4), transforming growth factor type α (TGFα, M r 5623, p I 6.8), insulin-like growth factor (IGF-I, M r 7649, p I 8.3), and basic fibroblast growth factor (bFGF, M r 15,000–17,000, p I 9.6) all transferred with high efficiency (74.1 ± 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46–98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% ( v v ) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGFα, and IGF-I were not detectable while 6.25–100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic p I proteins.