We studied several neurophysiological properties of in vitro maturing glycine receptors in mouse spinal cord neurons cultured for various times: 3-7 days (early), 10-12 days (intermediate), and 17-24 days (mature), using whole-cell and gramicidin-perforated techniques. The glycine-activated Cl- conductance increased about 6-fold during in vitro development, and the current density increased from 177+/-42 pA/pF in early to 504+/-74 pA/pF in mature neurons. The sensitivity to glycine increased transiently from 39+/-2.8 microM in early neurons to 29+/-1 microM in intermediate neurons. Using whole-cell recordings, we found that ECl did not change during development. With the gramicidin-perforated technique, on the other hand, ECl shifted from -27 to -52 mV with development. Thus, immature neurons were depolarized by the activation of glycine receptors, whereas mature neurons were hyperpolarized. The current decayed (desensitized) during the application of 500 microM glycine. The decay was single exponential and the time constant increased from 2,212+/-139 msec in early neurons to 4,580+/-1,071 msec in mature neurons. Picrotoxin (10 microM) inhibited the current to a larger extent in early neurons (46+/-6% of control), and the sensitivity of these receptors to strychnine (IC50) increased from 23+/-3 nM to 9+/-1 nM in mature neurons. In conclusion, several properties of spinal glycine receptors changed during in vitro neuronal maturation. This indicates that, similar to GABA(A) receptors, the functions of these receptors are developmentally regulated. These changes should affect the excitability of spinal neurons as well as other maturation processes.