This laboratory previously reported that zymosan priming protects rats against pulmonary oxygen toxicity. That study used a standard priming protocol (3 daily intravenous injections [15 mg zymosan/rat] with 2 days' rest) before hyperoxia. This study confirms that report and more fully characterizes the zymosan priming model. Three studies were conducted to establish the (1) effects of dosage, (2) role of duration of rest period between injections and hyperoxia and (3) importance of injection number, on protection by zymosan priming. Rats were exposed to >95% oxygen for 52 h or room air and acute lung injury was quantitated using standard methods. Lung injury decreased (P < 0.05 versus saline controls) in all groups of zymosan-primed rats (3 daily intravenous injections [1-15 mg zymosan/rat] with 2 days' rest before hyperoxia). Although the differences between zymosan-primed groups were not statistically significant, protection (as indicated by decreasing mean values of measured parameters of lung injury) increased with dosage. A one-day rest after injections was sufficient to partially protect zymosan-primed rats from hyperoxia (some measured parameters in the zymosan-primed group differed significantly from comparable values in the Saline group), but full protection (all measured parameters within a group differed significantly from Saline values) was not produced until rats received two days' rest before hyperoxia. Finally, one or two zymosan treatments produced partial protection against oxygen toxicity but three injections were needed to produce full protection. In conclusion, this study found that the standard priming protocol (3 zymosan injections with 2 days' rest before hyperoxia) was the most effective in protecting rats against hyperoxia.