Series 2 was the second series of Holby City. It consisted of 16 episodes which ran from 25 November 1999 before concluding on 9 March 2000.
- Anton Meyer, Cardiothoracic Consultant, Darwin - George Irving
- Kristie Collins, Senior House Officer / Cardiothoracic Surgical Registrar, Darwin - Dawn McDaniel
- Julie Fitzjohn, Ward Sister - Nicola Stephenson
- Victoria Merrick, Senior House Officer, Cardiothoracic Surgery, Darwin - Lisa Faulkner
- Jasmine Hopkins, Staff Nurse / Ward Sister, Darwin - Angela Griffin
- Karen Newburn, Ward Sister, Darwin - Sarah Preston
- Ray Sykes, Senior Staff Nurse, Darwin - Ian Curtis
- Nick Jordan, Cardiovascular Surgical Registrar, Darwin - Michael French (until episode 14)
- Mike Barratt, general surgical consultant - Clive Mantle
- Kath Shaughnessy, ward sister - Jan Pearson
- Danny Shaughnessy, healthcare assistant - Jeremy Edwards
- Tash Bandara, general surgical registrar - Thusitha Jayasundera
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