BACKGROUND To compare the inter-practice and inter-regional variability in laboratory test requests by general practitioners in Spain, through the measure of appropriateness indicators. METHODS A call for data was posted on the Redconlab website. We obtained production statistics for 2010 from laboratories in 37 different hospitals from diverse Spanish regions. The following appropriateness indicators were calculated: test requests per 1000 inhabitants, ratios of related tests requests and index of variability. The results obtained in the laboratories in the region of authors were compared to the rest of the participating laboratories in order to establish whether there were regional differences in the test requesting patterns. RESULTS The rate of request of the tests ranged from 31.5 per 1000 inhabitants for vitamin B12 to 372.6 per 1000 inhabitants for glucose. The index of variability ranged from 1.53 for glucose and triglycerides to 7.4 for alkaline phosphatase. Regarding the ratios of related test requests, the variability index ranged from 1.24 for folic acid/vitamin B12 to 26.38 for lactate dehydrogenase/alanine transaminase. The most frequently ordered tests were the ones with less variability, except for uric acid and urinalysis. No significant differences were identified between the results of the laboratories in the region of authors and the rest, except for urinalysis (p < 0.001), folic acid/vitamin B12 (p = 0.030), and transferrin/ferritin (p = 0.018). CONCLUSIONS A considerable variability exists in laboratory test ordering patterns by general practitioners across Spanish regions. Local habits must have been decisive as shown by the regional differences in the results of indicators of some tests. The study results bring out the need to accomplish interventions to improve appropriate use of laboratory tests.