Guidance of axon growth cones to their correct target cells is a fundamental step for establishing neural connections in the developing and regenerating nervous system. Developing axons are guided to their targets by extracellular guidance molecules. Recent studies show that most of these guidance molecules have bi-functional roles. They serve as either attractants or repellents, depending on the state of intracellular second messengers, i.e., Ca2+ and cyclic nucleotides, in nerve growth cones. The molecular mechanisms by which second messenger signaling is regulated in response to guidance molecules are largely unknown. Our laboratory studies these second messenger signaling mechanisms in axon guidance using developing and mature Xenopus spinal neurons in vitro. By combined approaches of nerve growth cone turning assays, Ca2+ imaging, electrophysiology, pharmacological and molecular manipulations, we analyze the behavior of growth cone responses and Ca2+ dynamics in response to guidance molecules, such as netrin-1 and Sema 3A. Our recent studies show that direct modulation of Ca2+ channels by cyclic nucleotide signaling in the plasma membrane (PM) and endoplasmic reticulum (ER) determines the polarity of bi-directional turning of a growth cone in response to netrin-1. We currently study the detailed molecular mechanisms underlying Ca2+ dynamics in growth cones resulting from Ca2+ entry through Ca2+ channels in the PM, Ca2+ release through internal Ca2+ channels in the ER and the modulation of these channels by cyclic nucleotide signaling during growth cone response to guidance signals. Furthermore, we are establishing a mature Xenopus spinal neuron culture system to compare second messenger signaling cascades in the mature nervous system. We hope that our studies of axon guidance signaling in developing and mature neurons will lead to a better understanding of the cellular and molecular mechanisms of the normal nervous system and will provide information leading to the design of drugs and treatments for nervous system abnormalities and injury.