Background: Low vitamin D levels had been reported to be common in normal subjects worldwide. Studies in the Middle East had reported extremely low levels of serum vitamin D, despite high exposure to sunlight. Aim: To estimate vitamin D serum concentration in a sample of apparently healthy subjects from Basrah, by 2 methods (chemiluminescent and fluorescent assays). Methods: The study was carried out on apparently healthy subjects during the period from September 2018 to February 2019. Quantitative determination of the total 25-hydroxy vitamin D in serum was made using chemiluminescent microparticle immunoassay and enzyme-linked fluorescent assay. Other parameters (calcium, phosphorus, alkaline phosphatase, parathyroid hormone, hemoglobin, and erythrocyte sedimentation rate and body mass index) were also measured. Results: The mean level of vitamin D measured by the two methods, was 11.57±6.63 ng/ml and 13.31±6.52 ng/ml by chemiluminescent and fluorescent assays respectively. The prevalence of vitamin D deficiency was high, where more than 80% of the 57 subjects had vitamin D level below 20 ng/ml. If the cut-off point of vitamin D deficiency was taken as 10 ng/ml, around 46% of the subjects were found deficient in both methods. Although the two methods of vitamin D assay were well correlated with each other, fluorescent assay gave, on average, a significantly higher levels compared with the chemiluminescent method. Serum parathyroid hormone, showed a negative correlation with vitamin D serum levels. After excluding children and females, no significant difference was found between adult smokers and non-smokers when vitamin D was measured by both methods.Conclusion: Vitamin D deficiency is common (> 80%) among normal subjects. The enzyme-linked fluorescent assay resulted in higher mean level than chemiluminescent assay. The use of a deficiency cut-off point of 10 ng/ml may be more appropriate.