Although calcium is thought to equilibrate across the nuclear envelope, the envelope nevertheless represents a significant barrier to rapid diffusion. We have investigated the delay to calcium diffusion caused by the nuclear envelope, and the effect of two agents on that delay: histone proteins, which will be actively transported by the pore complex, and wheat germ agglutinin, which inhibits active pore transport. Cultured sensory neurones of 12 day old chick embryos replated onto a polyornithine substrate on the day before experimentation displayed a simple ovoid shape without neurites. Immediately before experimentation nuclei were stained with 2 μM Hoechst 33342. The bathing medium was then switched to 120 mM NaCl, 1.2 mM MgCl₂, 5.5 mM KCl, 1.8 mM CaCl₂, 1.8 mM TEACl, 10 mM HEPES, 25 mM glucose, 1 µM tetrodotoxin and 5 µM (S)-(-)-BAY K 8644, pH 7.2. The presence of BAY K enhanced calcium currents and therefore increased the depolarization-evoked fluorescence signal. Neurons were whole cell patch clamped, at room temperature, on the stage of a Zeiss 510 confocal microscope through pipettes containing 125 mM CsCl, 4 mM MgATP, 10 mM HEPES and 100 µM of the calcium indicator Oregon Green BAPTA 488 Dextran, 10 kD, pH 7.2, plus 500 µg/ml of a histone mixture (Sigma H-7755) or 100 µg/ml of wheat germ agglutinin (Sigma L-9640) as appropriate. Neurones were held in whole cell mode for 3 min to allow diffusion of the dye and active agents into the cytosol and to the nuclear pores. Neurones were then scanned every 1.92ms along a line that passed through the nucleus (excitation 488 nm, emission >505 nm, confocal plane 3 µm). Depolarization (-70 mV to +10 mV for 50 ms) evoked a calcium increase that could be seen to pass from the cell edge to the cell centre within tens of milliseconds. Comparing the rising phase of the fluorescence signal in the nucleus, and at the same distance from the cell edge in the cytosol, yielded a value for the delay due to the nuclear envelope. In control cells this was 6.2 ± 2.7 ms (± SEM, N=10). In the presence of histone mixture the value was not significantly different (6.6 ± 4.7 ms, N=9). However, wheat germ agglutinin eliminated the delay (time difference -1.1 ± 2.1 ms, N=12). The last value is not significantly different from zero, but is significantly different (P<5%, t test) from the control value. Our tentative interpretation is that in control cells the majority of the nuclear pores are transporting macromolecular cargo, so that addition of extra cargo in the form of histone has little effect on the average pore topology. Wheat germ agglutinin inhibits the binding and transport of macromolecules so that the entire diameter of all the nuclear pores is available for calcium diffusion.